Spring 2011 Research Grant Recipients Talk About Their Research

Christopher Grigsby
Graduate Student

Abstract Title:
Oral Delivery of the Factor VIII Gene: Immunotherapy for Hemophilia A

BD: Tell us about your educational background.

Christopher Grigsby: I earned my undergraduate degree in bioengineering from the University of California, Berkeley, and worked as a diabetes researcher at the University of California, San Francisco. I am now a graduate student in biomedical engineering at Duke University.

BD: How did you become interested in science?

Christopher Grigsby: Like many researchers I have always been attracted to science and research. Research is a team effort, and I enjoy the synergy of ideas from group members. I've had great teachers and mentors since grade school, including my current research advisor. I believe that biomedical engineering, since it is applied, has the potential to make a more immediate difference in patients' lives than studying basic science.

BD: Please provide a broad overview of your research interests.

Christopher Grigsby: My general interest lies in non-viral gene delivery. Specifically, I'm studying polymer-based oral gene delivery. Unlike the most commonly known viral delivery methods, non-viral delivery does not alter the organism's genomic DNA or the basic genetic instructions of target cells. What attracts me to this methodology is that it's a platform technology that is broadly applicable to delivery of almost any gene.

BD: Please describe the project for which you were awarded the BD grant.

Christopher Grigsby: We're currently studying chitosan, a natural biopolymer found in the shells of crabs and insects, as the gene delivery vehicle. The gene-chitosan complex self-assembles because DNA is negatively charged and chitosan contains positively-charged domains. When test animals are administered such medicines orally the complexes adhere to cells in the gut, where they are taken up. The gene finds its way into the cell's nucleus—but not into the chromosomes—where it nevertheless instructs the cell to produce the therapeutic protein of interest. Furthermore, the production of the protein in the gut can signal the patient's immune system to tolerate the presence of that protein, which would otherwise be seen as foreign. We intend to use the system to deliver the factor VIII gene, which is absent in individuals with hemophilia A, both to correct the disease and abolish the inhibitory immune response to factor VIII.

This approach carries less risk of adverse events compared with conventional gene therapy, where the gene is introduced into the genome and passed down to progeny as the cell replicates. Here the effect is operative only as long as the cell survives, and dilutes through cell division. The only down-side is that patients must replenish the gene by re-introducing it.

BD: What are your short-term research goals?

Christopher Grigsby: We have two specific aims. The first is mechanistic and involves making the gene-chitosan nanoparticles, coating them in Eudragit®, a pH-sensitive polymer that is insoluble in the acidic environment of the stomach but dissolves at the higher pH in the intestine. Using a mouse model we will determine which regions and cells are assimilating the drug.

We then hope to test suitably engineered particles in a canine model, for which we already have some preliminary data. One dog used in a previous study had preexisting antibody inhibitors against the factor VIII protein, similar to the 30% of human patients who develop an immune response to the therapeutic protein. Interestingly, after being treated with the drug particles, this animal's inhibitors to the clotting factor became undetectable. We ascribe this finding to the fact that protein delivery to the mucosal immune system in the gut is tolerogenic. We're interested in testing whether this effect is reproducible.

BD: What are the long-term implications for human health?

Christopher Grigsby: Generating therapeutic proteins endogenously through oral gene therapy provides many advantages compared with injected protein drug delivery. Many patients who receive protein therapy, for example, develop inhibitory antibodies to these medicines. Immune responses toward self-generated proteins are much less likely.

The significance of an oral gene delivery platform cannot be overestimated. The dosage form is familiar to most patients, who are used to taking pills. More significantly, our approach is applicable to a variety of human diseases that involve the replacement or augmentation of a deficient protein, for example hemophilia and diabetes.

The drawback of polymer-based delivery is also its strength. Since cells are only transiently transfected the effect is temporary, meaning the patient must continue the treatment for life. On the other hand the risks of altering the genomes of cells, and all that viral transfection entails, are minimized.

BD: Which BD reagents do you plan to acquire?

Christopher Grigsby: We will purchase BD antibodies for flow cytometry to quantify the distribution of nanoparticles and the subpopulations of cells involved in therapeutic and tolerogenic responses. We'll also use ELISA to measure systemic responses.

Yael Korin, PhD
Associate Researcher of Pathology and Laboratory Medicine

Abstract Title:
Use of Immunophenotyping to Develop an Immune Profile of Tolerance in Pediatric Liver Transplant Recipients

BD: Tell us about your educational background.

Yael Korin: I received my undergraduate degree in genetics and zoology from the Hebrew University in Jerusalem. Afterward I studied for a master's degree in cell biology at California State University, Dominguez Hills, and for my PhD in virology and immunology at the University of California, Los Angeles. For my post-doctoral fellowship I remained at UCLA, where I still work.

BD: How did you become interested in science?

Yael Korin: Like so many scientists I grew up loving nature, animals, and plants. While still in high school I became attracted to biology as a formal discipline. From that point forward I knew I would become a biologist.

BD: Please describe your broad research interests.

Yael Korin: I am mostly interested in studying the role and function of the immune system, particularly the T- and B-cell adaptive immunity, in responses to pathology and disease. My interest in HIV, during graduate school, related to how T cells interact with the virus, and the fate of infected cells. Now I'm focusing on T-cell immunity as it applies to organ transplantation and rejection.

BD: Describe the project for which you were awarded the BD grant.

Yael Korin: Immunosuppressive drugs have dramatically improved the prospect for recipients of transplanted organs. But the drugs are expensive, have side effects—some quite serious—and usually must be taken for life.

But the need for immunosuppression varies greatly among transplant recipients, particularly those receiving livers. About 20% of these individuals are immune tolerant, meaning they thrive without immunosuppression. The ability to identify tolerant patients who can maintain graft acceptance in the absence of immunosuppression is therefore a highly desirable goal in liver transplantation. Equally important is the ability to determine which patients are non-tolerant and cannot be safely withdrawn from immunosuppression.

In the proposed project, we will employ immunophenotyping to characterize peripheral blood lymphocytes of tolerant liver transplant recipients and to compare tolerant patients to those who are non-tolerant and to stable pediatric liver transplant recipients. We hypothesize that immunophenotyping of peripheral blood lymphocytes will help define an immune profile of tolerance that can be used to identify which children are candidates for minimization or complete withdrawal of immunosuppression.

BD: What are your short-term research goals?

Yael Korin: We hypothesize that the composition of the transplant recipient's T cells in the naïve, effector, or regulatory cell compartment will have either pro-inflammatory or pro-tolerogenic effects on allograft acceptance and survival. Our first aim will be to analyze and compare peripheral blood lymphocyte subsets in tolerant, non-tolerant, and stable pediatric liver transplant recipients. We will use multiparameter flow cytometry to assess the frequency of naïve, central memory, effector memory, and regulatory and suppressor cells. Our second aim is to relate each recipient's peripheral cell phenotype to clinical outcome, including allograft function and biopsy histology, to develop immune profiles that characterize tolerance and non-tolerance.

BD: What are the implications of your work for human health?

Yael Korin: The quantitative analysis of a transplant recipient's T- and B-cell repertoire can be used to assess rejection risk and aid immunosuppressive regimen design. We expect that data from these studies will help to identify patients most likely to benefit from early aggressive immunosuppressive therapy or, alternatively, those who may be candidates for drug-sparing protocols.

BD: Which BD reagents to you expect to use?

Yael Korin: We will conduct multiparameter flow cytometric immunophenotyping with 9-color monoclonal antibody panels conjugated to FITC, PE, PerCP, PerCP-Cy™5, APC, APC-H7, PE-Cy™7, Pacific Blue™, and BD Horizon™ V450. For cell fluorescence we use a BD™ LSR II flow cytometer from BD Biosciences.

Doris Lambracht-Washington, PhD
Assistant Instructor of Neurology

Abstract Title:
Phenotypic and Functional Characterization of T Cell Subsets Generated in DNA Aβ42 Trimer Immunized Mice to Determine Safety in Immunotherapy for Alzheimer's Disease

BD: Tell us about your educational background.

Doris Lambracht-Washington: I received my bachelor's and master's degrees from the Technical University in Hannover, Germany. My undergraduate diploma was in zoology, genetics, immunology, and biochemistry. For my master's in molecular biology I worked on light-induced microRNA in Pisum sativum, the green pea. I completed my doctorate in immunology and genetics at the Medical University of Hannover, where I studied non-classical major histocompatibility complex (MHC) class I genes. During my post-doctoral fellowship at the University of Texas Southwestern Medical Center I analyzed non-classical or class Ib MHC genes in mouse and rat, which involved basic immunogenetics.

BD: How did you become interested in science?

Doris Lambracht-Washington: Most children like nature, animals, flowers, plants, and I was no exception. At one time I wanted to be a veterinarian, but by high school I became fascinated by genetics and immunology, so my decision to become a basic scientist was easy.

BD: Please provide a broad overview of your research interests.

Doris Lambracht-Washington: If I have to summarize it in one word, it would be "immunology." How cells interact during immune responses fascinates me, for example how they communicate via cytokines and chemokines, and how they become activated. While this is a fast-developing field we actually know very little about it. There are so many subtle interactions and connections. I'm particularly interested in how autoimmune responses arise in the brain, and why they cause so much inflammation and neurodegeneration.

BD: Describe the project for which you were awarded the BD grant.

Doris Lambracht-Washington: Our group is developing a vaccine, for use in Alzheimer's disease (AD) patients, against amyloid beta 1-42 (Aβ42). This neurotoxic component of plaques that are the hallmark of AD destroys the brain and in particular the hippocampus, which is the seat of memory. We are currently using a gene gun vaccine delivery system in mouse models of AD. Genetic immunization, which involves injecting the gene encoding the antigen, causes a very different immune response from injection of the peptide antigen itself. We are investigating this delivery mode because an earlier human study employing peptide-based antigen generated an antibody immune response and T-cell-mediated inflammation as well. A few patients developed encephalitis and the trial was halted.

In our method, once the gene is inside cells it migrates to the nucleus, where it instructs the cell to produce the Aβ42 peptide. In our mouse model we propose that an inflammatory T-cell response will not occur due to a T-helper 2 immune response, which appears to dampen at later immunization time points. We still have to show that inflammatory T cells are absent in the brain, but it is clear that immunization reduces plaque buildup in the brain and the animals retain their ability to learn new tasks.

BD: What are your short-term goals?

Doris Lambracht-Washington: We will examine a possible regulatory T-cell response in mice immunized with the Aβ42 gene. It makes sense that, in an immune response against a self-antigen, regulatory T cells play an important role. We expect to see different T-cell markers, activation markers, cytokines, cytokine activation, and T-cell subsets in DNA-immunized vs peptide-immunized mice. In a different project we are collaborating with groups that will provide functional and behavioral assessment of the vaccine's effectiveness to see whether the treated mice have a better memory and do not forget what they have learned. While we expect to see less plaque in test animals' brains, it is even more important to observe such functional improvements.

BD: What are the long-term implications for human health?

Doris Lambracht-Washington: Almost everybody has a friend or family member with AD, for which no cure or effective treatment exists. Within a few years we hope to conduct human clinical studies on a vaccine similar to the one we are working on now in mice. If mouse studies are any indication, the vaccine could be used to prevent AD in individuals whose families have a high predisposition toward developing AD. Such a vaccine might also prevent progression of the disease.

BD: What BD reagents to you expect to acquire with your grant?

Doris Lambracht-Washington: I have a long list of antibodies for studying T cells and cytokine markers. We will also use kits for cell isolation and characterization through flow cytometry, as well as basic reagents for immunology and cell proliferation.

Dean Lee, MD, PhD
Assistant Professor

Abstract Title:
Expanded NK Cells for Adoptive Immunotherapy

BD: Tell us about your educational background.

Dean Lee: I received my BA degree in natural sciences from Fresno Pacific College, which has since changed its name to Fresno Pacific University. I then entered a PhD program in molecular biology, with an emphasis on immunology, at Loma Linda University. Two years later I was accepted into an MD program at Loma Linda.

BD: How did you become interested in science?

Dean Lee: Science has been my primary interest since grade school. I grew up in a small town in northern California, where they had a unique program for gifted minors. Through that program I got to do science projects on my own and get a feel for the scientific method. By secondary school I thought I'd go into biomedical engineering, but settled on pursuing medicine and medical research.

BD: Please provide a broad overview of your research interests.

Dean Lee: If I had to sum it up in one phrase, it would be translational medicine focusing on natural killer (NK) cell therapy. Until a few years ago many biologists avoided these cells due to their complexity. That's changing rapidly. Unlike T cells, which require prior exposure to specific targets, NK cells operate more broadly, with generalized activity against many cancers, for example lymphoid tumors, carcinomas, and sarcomas. T cells are like the FBI looking for a particular most-wanted person, whereas NK cells function more like the border patrol.

Adoptive transfer of NK cells—the transfer of these lymphocytes from a healthy donor to a patient—has been shown to be safe in adult cancer patients, which makes these cells suitable for investigation as cancer therapies. The downside is that unlike T cells, NK cells are difficult to grow in the lab in therapeutically relevant numbers. Our lab focuses on creating artificial engineered helper cells that stimulate NK cells to grow. We're examining one of those along with interleukin (IL)-21.

BD: Describe the project for which you were awarded the BD grant.

Dean Lee: Propagating NK cells in vitro is confounded by the many activating and inhibitory receptors, cooperative receptor pairs, and overlapping signaling pathways for maturation, activation, and proliferation. Moreover, the expanded cells are limited by senescence due to telomere shortening.

Up to now most research in this area utilized IL-15 as the primary signal for NK cells. Our lab is examining IL-21 for this purpose. Previous groups had shown 277-fold expansion using IL-15. We have archived expansion of 23,400-fold by engineering antigen-presenting cells to express membrane-bound IL-21, which signals through the telomerase activator STAT3. This produces enough material for multiple, high-dose infusions of NK cells. My institution is conducting four clinical trials on various cancers using adoptive transfer of NK cells.

We hope to uncover the role of IL-21 in regulating telomerase expression. Using STAT inhibitors and STAT-specific BD Phosflow™ analysis, we will determine the relative importance of STAT3 and STAT5 phosphorylation in regulating telomerase expression in NK cells.

Our lab will also attempt to clarify which NK cell subpopulations are involved in IL-21-dependent expansion, the role of IL-21 in regulating the major histocompatibility complex binding protein CD160, and how together, IL-21 and CD160 regulate NK cell survival.

BD: What are the short-term goals of your work?

Dean Lee: Our immediate goal is to understand the activity of STAT3 in NK-cell expansion. Some mouse data suggests that STAT3 is not important for NK cells; why it might be significant in human NK cells will help us understand how human and mouse immunity differ. And ultimately, we would like to know what genes are being activated in cells produced for therapy, and why the NK cells are longer-lived.

BD: What are the long-term implications for NK cell-based immunotherapy?

Dean Lee: The literature tells us that NK function predicts cancer survival in all major therapeutic settings. So the ultimate goal is to improve NK cell-based immunotherapy. To achieve that, we need to learn how to enhance NK-cell function.

We're working on both allograft and autograft protocols for solid tumors and lymphoma. NK cells expanded ex vivo and administered as autografts do not cause damage to normal tissues. And however dysfunctional they might have been inside the body, expansion helps them recover their anti-tumor activity. Another benefit is that these cells have high expression of the CD16 antibody receptor. If we can augment the efficacy of an anti-cancer antibody by providing more NK cells that work through the antibody, then we can make those therapies even better.

But most NK cell trials have been allografts. A seminal paper in Blood by Jeff Miller at the University of Minnesota suggests that adoptive transfer of NK cells from properly selected donors can induce remissions. Understanding how STAT3 works could significantly improve prospects for these patients.

BD: What BD reagents do you plan on acquiring?

Dean Lee: BD™ Cytometric Bead Array (CBA), apoptosis reagents, and STAT BD Phosflow™ reagents immediately come to mind.

Flavia Pereira, PhD
Post-Doctoral Fellow

Abstract Title:
Role of CD13 in Wound Healing after Myocardial Infarction

BD: Tell us about your educational background.

Flavia Pereira: I received my undergraduate degree in pharmacy at the University Department of Chemical Technology, Mumbai, India, and my PhD in pharmacology and pharmaceutical sciences at the University of Montana, Missoula. At Montana my research involved the relationship between arsenic in ground water and atherosclerosis. Following graduate school I continued research as a post-doctoral fellow at Temple University and the University of Connecticut Health Center, where I continued to study cardiovascular diseases.

BD: How did you become interested in science?

Flavia Pereira: I've known since high school that I wanted to study biology, but I was unsure whether to the best way to do this was as a medical doctor or a basic scientist. Later, while pursuing my bachelor of pharmacy degree, I became attracted to pharmacology, which I eventually combined with my interest in cardiovascular biology.

BD: Please provide a broad overview of your research interests.

Flavia Pereira: The thrust of my research today is myocardial infarction and atherosclerosis. Although they're technically different diseases, they're related, since one can lead to another. The principal target of my current research is CD13, a large, cell surface peptidase expressed on monocytes and macrophages. CD13 is significantly upregulated on endothelial cells and monocytes at sites of inflammation. The role of CD13 in inflammatory diseases remains largely unexplored.

BD: Please describe the project for which you were awarded the BD grant.

Flavia Pereira: Following a heart attack, or MI, injured tissue heals through the interdependent processes of inflammation, scar formation, and tissue remodeling. The inflammatory phase following MI depends on the migration of monocytes and fibrocytes to the site of injury. This is mediated by the adhesion molecules that are upregulated on the activated endothelial cells lining the vessels of damaged tissue. After transmigrating through the endothelial barrier and into tissue, monocytes differentiate into macrophages that produce TGF-ß1. This growth factor causes resident fibroblasts to differentiate into myofibroblasts, which in turn generate extracellular matrix proteins. The relative contributions of each of the component cell types to the healing process, the precise mechanisms regulating cell differentiation, and the processes that control the trafficking of circulating cells are not well understood.

Our preliminary studies showed that CD13 functions as an adhesion molecule and mediates monocyte/endothelial interactions critical to inflammatory trafficking. CD13 is expressed on the macrophages and endothelial cells as well as on myofibroblasts within the infarct. After myocardial infarction, CD13-null mice have lower cardiac output, thinner ventricular walls, reduced numbers of infarct-resident macrophages and myofibroblasts, and decreased collagen deposition. We propose that adverse cardiac remodeling in these mice results from defective trafficking and/or function of these cells in the healing heart.

To test our hypothesis, we will induce MIs in wild type and CD13-null mice and analyze the role of CD13 in monocyte and fibrocyte trafficking to the injured heart. We will also determine whether the function of macrophages and myofibroblasts is altered due to lack of CD13, resulting in adverse cardiac remodeling following myocardial infarction.

BD: What are the short-term goals of your work?

Flavia Pereira: To determine the contribution of CD13 expressed on monocytes and myofibroblasts in inflammation and remodeling post-MI. In addition to the global CD13 knockout mice, we are developing a fibroblast specific CD13 knockout mouse model to look specifically at those cells.

BD: What are the implications for human health?

Flavia Pereira: The role of CD13 in cardiovascular diseases is unknown. A great deal of work remains before one could consider clinical applications. However, recent studies have shown that CD13 can be used as a receptor for targeting peptides in vivo. Eventually, one could imagine CD13 as a drug therapy target.

BD: What BD reagents do you expect to acquire through your grant?

Flavia Pereira: We do a lot of multicolor flow cytometry in our lab using the BD™ LSR II and BD FACSCalibur™ instruments. We'll need three or four markers to identify our target cell populations, plus reagents, antibodies such as CD11b, CD3, CD19, NK1.1, LY6G, and CD11c, and magnetic beads for enriching cell populations.

Richard Robinson, PhD
Assistant Professor

Abstract Title:
Influence of Soluble IL12Rβ1 on TH1/TH17 Differentiation

BD: Tell us about your educational background.

Richard Robinson: I received my BS degree in biology from Anderson College, a small liberal arts school in Anderson, South Carolina. After graduation, I worked as a technician at the University of South Carolina School of Medicine. I received my PhD in microbiology and immunology from Dartmouth College. During my postdoctoral fellowship, at the Trudeau Institute, I studied mouse models of infectious diseases.

BD: How did you become interested in science?

Richard Robinson: I was a relatively late bloomer. I originally intended to major in religion or philosophy. However, my biology professors and positive undergraduate research experiences inspired me to become a biologist. I was also greatly influenced by the writings of the late Carl Sagan.

BD: What are your broad research interests?

Richard Robinson: I'm interested in understanding how the immune response controls infection with the tuberculosis bacterium. We use both a mouse model of this disease as well as responses of human peripheral blood immune cells. We've made several observations in a mouse model that we believe will translate to human infection. I hope, eventually, to understand how immunity affects tuberculosis as well as other bacterial infections.

BD: Describe the project for which you received the BD Grant.

Richard Robinson: We're focusing on IL (interleukin) 12Rβ1, a receptor generated by dendritic cells that spans the cell membrane and binds to the p40-domain of IL-12 and IL-23, and IL homodimer. These cytokines broadly affect immune responses involved in both pathogen-driven immunity and autoimmunity. The association of IL12Rβ1/cytokine complexes with co-receptors IL12Rβ2 or IL23R confers cytokine specificity and initiates downstream signaling cascades.

We discovered earlier that an isoform of IL12Rβ1 lacking a transmembrane domain contains an altered C-terminus. Despite these differences the protein, which we named soluble IL12Rβ1 (sIL12Rβ1), retains the ability to associate with the cell membrane, binds radiolabeled-IL12p40, and enhances the effects of IL12p40-homodimer on dendritic cells.

We will study how sIL12Rβ1 influences the differentiation of T lymphocytes into T-effector lymphocytes, an important player in immunologic responses that produces infection-fighting proteins. The BD grant supports work on mice and in vitro studies in human peripheral blood lymphocytes.

BD: What is the relationship between IL12Rβ1 and tuberculosis?

Richard Robinson: Multiple genetic association studies support the idea that individuals deficient in IL12Rβ1 experience more severe or more persistent cases of tuberculosis. Interestingly, IL12Rβ1-deficient individuals also have a limited ability to control the live BCG tuberculosis vaccine.

BD: What are the short-term goals of your work?

Richard Robinson: Using antibodies we've developed in our lab, we will test if neutralizing sIL12Rβ1 affects how T cells differentiate. We hypothesize that they will fail to differentiate into protective T cells in our mouse model. We will also study how this affects signaling pathways known to promote T-cell development.

BD: What are the long-term implications of this work for human health?

Richard Robinson: Assuming sIL12Rβ1 broadly affects immune responses, one can envision two therapeutic strategies involving this protein. Inactivating sIL12Rβ1 with an appropriate antibody could potentially reduce the clinical manifestations of autoimmune diseases. A more challenging approach involves increasing the activity of sIL12Rβ1 to fight infection.

BD: What BD reagents do you plan to use in your work?

Richard Robinson: We'll use antibodies for staining peripheral blood cells and cell sorting with BD flow cytometers. ELISA reagents will also come in handy for quantifying interferon gamma release. Finally, we are interested in antibodies for studying the phosphorylation of intracellular proteins.

Michael Sheard, PhD
Director, Flow Cytometry Core Facilities

Abstract Title:
Effect of Lenalidomide on the Anti-Neuroblastoma Cytotoxicity of Human Natural Killer Cells Expanded from Blood Using K562-IL21 Feeder Cells

BD: Tell us about your educational background.

Michael Sheard: I received my undergraduate degree from East Carolina University in Greenville, North Carolina, with a double major in music and biology. After working at the National Institutes of Health I traveled to the Czech Republic, where I earned my PhD in oncology from Charles University in Prague. I spent time as a post-doctoral researcher at Masaryk Memorial Cancer Institute in Brno, also in the Czech Republic. Upon returning to the United States I did a senior post-doc at Children's Hospital Los Angeles, where I worked on the immune response against neuroblastoma.

BD: How did you become interested in science?

Michael Sheard: I began my academic career as a music major. At one point during college I developed a strong desire to work in some area of oncology. That's when I added my second major in biology.

BD: Please provide a broad overview of your research interests.

Michael Sheard: I'm interested in harnessing the immune system to fight cancer. Right now I'm working on natural killer cells, learning how to expand their populations to clinically relevant numbers and then activate their cytolytic function.

BD: Describe the project for which you were awarded the BD grant.

Michael Sheard: Cellular immunotherapy holds the potential for treating cancer with minimal side effects. Natural killer (NK) cells, in particular, are known to bind to and kill cancer cells despite the tumor's low expression of major histocompatibility complex (MHC) class 1 proteins, which the immune system uses to recognize "self" from "non-self." Neuroblastoma, a pediatric cancer, is therefore a good target for NK cell therapy because it rarely expresses detectable levels of MHC class 1 proteins.

While it's possible to harvest NK cells from human blood, their numbers are quite small and expanding them to highly functional killers is complicated. We have found that a two-week incubation of peripheral blood mononuclear cells with "feeder cells"—actually a human leukemia cell line that expresses interleukin-21 on their cell surface—expands the NK cells approximately 2,000-fold. B cells and monocytes, which are undesirable, are virtually absent from the final product. Moreover, the expanded NK population expresses surface receptors, among them NKG2D, DNAM-1, and CD56, that promote tumor cell recognition.

BD: Where does lenalidomide fit in?

Michael Sheard: Lenalidomide, a drug approved for multiple myeloma, has beneficial effects on anti-cancer immune responses, specifically by helping to expand and activate NK cells. We know it works in clinical trials on non-transplanted NK cells, and hope it will be similarly effective for expanded cells.

BD: What are your near-term scientific objectives?

Michael Sheard: At this point we can obtain a few million NK cells from the blood and expand them to billions, which may represent sufficient numbers to re-introduce therapeutically. The possibility that lenalidamide treatment can make these cells even more effective is exciting. So we're planning to examine the phenotype, functional status, and therapeutic efficacy of NK cells expanded ex vivo with feeder cells and activated in vivo with lenalidomide. Our analytic methods will include cytometric bead array assays, polychromatic immunophenotyping panels, and established cytotoxicity assays.

BD: What are the implications of your work for human health?

Michael Sheard: Eventually we hope to test ex vivo-expanded NK cells in neuroblastoma patients, in combination with infusions of lenalidomide. Accumulating evidence suggests that approaches employing NK cells can succeed. For example, a recently completed antibody treatment improves survival in stage 4 patients by 20%. This antibody binds to GD2 molecules that are abundant on neuroblastoma cells, and NK cells are believed to ligate to the bound antibody and subsequently mediate the death of the neuroblastoma cells.

BD: What BD reagents and supplies do you expect to purchase with your grant money?

Michael Sheard: Because there are many activation states of NK cells, lymphocytes, and monocytes, we routinely use multiparameter flow cytometry to study these cells, employing 10-color staining to examine each cell individually. So to carry out this work we’ll need approximately 30 fluorescently-labeled antibodies. In addition I expect to use the BD™ Cytometric Bead Array, a very exciting technology, to quantify soluble proteins in blood and in fluids from in vitro cell cultures. These reagents should allow my colleagues Bob Seeger, Yibing Xu, Yin Liu, and me to answer many of this project’s remaining questions.