SILVERSTEIN, SAMUEL C., M.D. Emeritus John C. Dalton Professor of Physiology & Cellular Biophysics, and Professor of Medicine Structure and functions of polymorphonuclear (PMN), mononuclear phagocytes, lymphocytes, platelets and endothelial cells (EC) in innate immunity and diseases. Email:scs3@columbia.edu

Current Research

Cellular Immunotherapy: My colleagues and I have developed a quantitative, experimentally verified model for cellular immunotherapy of neoplastic and infectious diseases. The model incorporates an equation derived previously to describe neutrophil bactericidal activity in three dimensional fibrin gels in vitro and in sub-cutaneous bacterial infections in vivo. As with neutrophils, we have found that the concentration of antigen-specific human and mouse CD8+T-cells determines their efficacy in killing cognate antigen–expressing target cells in two dimensional tissue culture plates, in three dimensional collagen-fibrin gels, and in established melanomas in vivo.

Using limiting dilution assays we have found that only 2-2.8% of CD8+T-cells are cytolytically active, and that the cytolytically active CD8+T-cells account for all tumoricidal activity of the entire CD8+T-cell population. Cytokines, such as IL-15 and IL-21 and adjuvants such as anti-CD40, in combination with IL-2, increase the fraction of cytolytically active CD8+T-cells in a population. Quantitative analyses of these cytokine-activated CD8+T-cells show that CD8+T-cell activation in the presence of specific cytokines and/or adjuvants increases not only the percentage of cytolytically active CD8+T-cells in a population, but also the specific cytolytic activity of each T-cell. Thus, activation of CD8+T-cells in the presence of cognate antigen produces an increase in the number of antigen-specific CD8+T-cells, while activation of CD8+T-cells in the presence of antigen plus adjuvant/cytokines produces increases in both the number and quality of the cytolytic CD8+T-cells. Thus we have been able to separate, quantitatively, the effect of antigen vs. adjuvant on the quantity and quality of CD8+T-cells.

The three dimensional collagen-fibrin gel assays we employ mimic precisely the efficiency with which antigen-specific CD8+T-cells kill cognate antigen-expressing target cells in mouse spleen in vivo. Accordingly, they can be used as a “standard” for the cytolytic potential of any preparation of tumor antigen-specific CD8+T-cells. By comparing the tumoricidal activity of a given CD8+T-cell preparation in vitro with its tumoricidal activity in vivo, we can obtain a measure of the aggregate immunosuppressive activity of the intra-tumoral environment. Our studies show it is about 50% for B16 mouse melanoma.

The equation that models CD8+T-cell killing of cognate antigen-expressing target cells (bt = b0 e-kpt + gt, in which bt is the concentration of antigen-expressing target cells at time t, b0 is the concentration of antigen-expressing target cells at t = 0, k is an experimentally derived value that defines the rate of killing of antigen-expressing target cells in ml/cognate antigen-specific CD8+T-cell/min, p is the concentration of cognate antigen-specific CD8+T-cells, and g is the growth rate of antigen-expressing target cells/min), enables us to calculate the critical intra-tumoral T-cell concentration (CTC) of total and cytolytically active antigen-specific CD8+T-cells that must be achieved to control tumor growth. For B16 melanomas in syngeneic mice these concentrations are 3 x 106 total and 6 x 104 cytolytically active antigen-specific CD8+T-cells/g tumor.

Our plans for the future are to isolate populations of cytolytically active CD8+T-cells using available high throughput single cell analysis and isolation technologies, and to use these cells to answer the following questions :

1. Is expression of cytolytic activity a stochastic or clonal property of CD8+T-cells?

2. What are the molecular characteristics of cytolytically active CD8+T-cells that distinguish them from their cytolytically inactive siblings?

3. Can we identify a plasma membrane marker that will enable us to identify and select the cytolytically active cells in a CD8+T-cell population? Alternatively, can we identify a plasma membrane marker that will enable us to identify and selectively remove the cytolytically inactive cells in a CD8+T-cell population?

4. Can we identify methods to induce 10-50% of CD8+T-cells in a population to express cytolytic activity?

5. Will populations of antigen-specific CD8+T-cells containing 10% or more cytolytically active antigen-specific CD8+T-cells produce sterilizing immunity against melanomas in mice and humans, as our calculations and studies suggest?

Bibliography:

1. Li, Y., Loike, J.D., Ember, J.A., Cleary, P.P., Lu, E., Budhu, S., Cao, L., and Silverstein, S.C. The bacterial peptide N-formyl-Met-Leu-Phe inhibits killing of Staphylococcus epidermidis by human neutrophils in fibrin gels. J. Immunology 168(2):816-24, 2002.

Li, Y., Karlin, A., Loike, J.D., Lu, E. and Silverstein, S.C. A Critical Concentration of Neutrophils is Required for Efficient Bacterial Killing in Suspension. Proc. Natl. Acad. Sci. U.S.A., 99(12):8289-8294, 2002.

2. Li, Y., Karlin, A., Loike, J., and Silverstein, S.C. A critical concentration of neutrophils is required to block growth of S. epidermidis in fibrin gels. J. Exp. Med. 200:613-622, 2004.

3. Budhu, S., Loike, J.D., Pandolfi, A, Han, S., Catalano, G., Constantinescu, A., Clynes, R. and Silverstein, S.C. CD8+ T-cell concentration determines their efficiency in killing cognate antigen-expressing syngeneic mammalian cells in vitro and in tissues. J. Exp. Med. 207(1):223-235, 2010.

4. Ganusov, V.V., Barber, D.L., and De Boer, R.J. Killing of targets by CD8+T-cells in the mouse spleen follows the law of mass action. PLoS ONE. 6(1), pp. 1-8, 2011 4. Silverstein, S.C. and Rabadan R. How many neutrophils are enough (redux, redux)? J. Clinical Investigation122(8):2776-9, 2012, 2012.

5. Emens, L.A., Silverstein, S.C., Khlief, S. Marincola, F.M., and Galon, J. Toward integrative cancer immunotherapy: targeting the tumor microenvironment. J. Translational Medicine 10:70, pp. 1-5, 2012.

Selected Publications

Reich-Slotky, R., Kabbash, C., Della-Latta, P., Blanchard, J.S., Feinmark, S.J., Freeman, S., Kaplan, G., Shuman, H.A., and Silverstein, S.C. Gemfibrozil (Lopidtm) Inhibits Legionella Pneumophila and Mycobacterium Tuberculosis Enoyl-Coa Reductases and Blocks Intracellular Growth of these Bacteria in Macrophages

Li, Y., Karlin, A., Loike, J.D., and Silverstein, S.C. 2004. Determination of the critical concentration of neutrophils required to block bacterial growth in tissues. J Exp Med. 200(5):613-22.

Loike, J.D., Shabtai, D.Y., Neuhut, R., Malitzky, S., Lu, E., Husemann, J., Goldberg, I.J., and Silverstein, S.C. 2004 Statin inhibition of Fc receptor-mediated phagocytosis by macrophages is modulated by cell activation and cholesterol. Arterioscler Thromb Vasc Biol. (11):2051-6.

Wyss-Coray , T., Loike, J.D., Brionne, T.C., Lu, E., Anankov, R., Yan, F., Silverstein, S.C., and Husemann, J. 2003. Adult mouse astrocytes degrade amyloid-beta in vitro and in situ.
Nat Med. (4):453-7

Husemann, J., Loike, J.D., Anankov, R., Febbraio, M., Silverstein, S.C. 2002. Scavenger receptors in neurobiology and neuropathology: their role on microglia and other cells of the nervous system. Glia. (2):195-205. Review

Li, Y., Karlin, A., Loike, J.D., Silverstein, S.C. 2002. A critical concentration of neutrophils is required for effective bacterial killing in suspension. Proc Natl Acad Sci U S A. (12):8289-94.

Berger, M., Budhu, S., Lu, E., Li, Y., Loike, D., Silverstein, S.C., Loike, J.D. 2002. Different G(i)-coupled chemoattractant receptors signal qualitatively different functions in human neutrophils. J Leukoc Biol. May;71(5):798-806.

Secondary Science Education:

My concerns about the quality of secondary science education in U.S. public schools led me to found Columbia University’s Summer Research Program for Science Teachers. The program, now in its 25th year, enables science teachers to engage in hands-on laboratory research under the mentorship of Columbia University faculty for 16 weeks in two consecutive summers and to participate in weekly professional development during this period. Over 300 secondary science teachers from the greater New York metropolitan area and their more than 250,000 students have benefited from this program since its inception. The program’s fundamental premise is that it is difficult to teach well something one has never done. Few science teachers have been challenged to use the concepts and tools of contemporary science to investigate a scientific question. Accordingly, they find it difficult to teach science in a guided inquiry-driven, hands-on manner. Columbia’s program addresses this problem. Accordingly, pre-post program studies document that 10-20% more students of teachers who have completed the program passed a New York State science Regents exam than did so in the year prior to the teacher’s entry into the program, or than students of other teachers in the same school teaching the same science subject. The program has been recognized by the New York City Mayor’s Award for Public Understanding of Science and Technology (2003) and the American Society for Cell Biology’s Bruce Alberts Award for Excellence in Science Education (2005), and replicated by Stanford University and the Ministry of Education of Singapore.

Teachers work in all disciplines of science represented at Columbia University (biology, medical sciences, psychology, chemistry, physics, astronomy, earth sciences, ecology, engineering, etc.), and are supervised and mentored by the faculty member who heads the laboratory to which they are assigned. One day per week is reserved for formal professional development exercises. The professional development day curriculum emphasizes such topics as science vocabulary, size and scale, modeling earth, physical, and biological science processes, and data driven instruction. For interested graduate students and post-doctoral fellows, it provides opportunities to mentor and work directly with middle and high school science teachers in the summer and/or during the academic year, and to engage in cutting edge research in science education. Students interested in learning more about the program and the opportunities it presents should contact Dr. Silverstein.

1. Wenglinsky, H. and Silverstein, S.C. The Science Training Teachers Need. Educational Leadership 64 (4):24-49, 2006-07

2. Silverstein, S.C., Dubner, J., Miller, J., Glied, S. and Loike, J.D. Teachers’ Research Program Participation Improves Their Students’ Achievement in Science. Science 326:440-442, 2009.