FlowKit: A Python Toolkit for Integrated Manual and Automated Cytometry Analysis Workflows.

Thomas N. Denny, MSc, M.Phil, is the Chief Operating Officer of the Duke Human Vaccine Institute (DHVI), Associate Dean for Duke Research and Discovery @RTP, and a Professor of Medicine in the Department of Medicine at Duke University Medical Center. He is also an Affiliate Member of the Duke Global Health Institute. Previously, he served on the Health Sector Advisory Council of the Duke University Fuquay School of Business. Prior to joining Duke, he was an Associate Professor of Pathology, Laboratory Medicine and Pediatrics, Associate Professor of Preventive Medicine and Community Health and Assistant Dean for Research in Health Policy at the New Jersey Medical School, Newark, New Jersey. He has served on numerous committees for the NIH over the last two decades and currently is the principal investigator of an NIH portfolio in excess of 65 million dollars. Mr. Denny was a 2002-2003 Robert Wood Johnson Foundation Health Policy Fellow at the Institute of Medicine of the National Academies (IOM). As a fellow, he served on the US Senate Health, Education, Labor and Pensions Committee with legislation/policy responsibilities in global AIDS, bioterrorism, clinical trials/human subject protection and vaccine related-issues.

As the Chief Operating Officer of the DHVI, Mr. Denny has senior oversight of the DHVI research portfolio and the units/teams that support the DHVI mission. He has extensive international experience and previously was a consultant to the U.S. Centers for Disease Control and Prevention (CDC) for the President’s Emergency Plan for AIDS Relief (PEPFAR) project to oversee the development of an HIV and Public Health Center of Excellence laboratory network in Guyana. In September 2004, the IOM appointed him as a consultant to their Board on Global Health Committee studying the options for overseas placement of U.S. health professionals and the development of an assessment plan for activities related to the 2003 PEPFAR legislative act. In the 1980s, Mr. Denny helped establish a small laboratory in the Republic of Kalmykia (former Soviet Union) to improve the care of children with HIV/AIDS and served as a Board Member of the Children of Chernobyl Relief Fund Foundation. In 2005, Mr. Denny was named a consulting medical/scientific officer to the WHO Global AIDS Program in Geneva. He has also served as program reviewers for the governments of the Netherlands and South Africa as well as an advisor to several U.S. biotech companies. He currently serves as the Chair of the Scientific Advisory Board for Grid Biosciences.

Mr. Denny has authored and co-authored more than 200 peer-reviewed papers and serves on the editorial board of Communications in Cytometry and Journal of Clinical Virology. He holds an M.Sc in Molecular and Biomedical Immunology from the University of East London and a degree in Medical Law (M.Phil) from the Institute of Law and Ethics in Medicine, School of Law, University of Glasgow. In 1991, he completed a course of study in Strategic Management at The Wharton School, University of Pennsylvania. In 1993, he completed the Program for Advanced Training in Biomedical Research Management at Harvard School of Public Health. In December 2005, he was inducted as a Fellow into the College of Physicians of Philadelphia, the oldest medical society in the US.

While living in New Jersey, Mr. Denny was active in his community, gaining additional experience from two publicly elected positions. In 2000, Mr. Denny was selected by the New Jersey League of Municipalities to Chair the New Jersey Community Mental Health Citizens’ Advisory Board and Mental Health Planning Council as a gubernatorial appointment.

The Weinhold Laboratory is currently focused on utilizing a comprehensive repertoire of highly standardized and formerly validated assay platforms to profile the human immune system in order to identify immunologic signatures that predict disease outcomes. These ongoing studies span a broad range of highly relevant clinical arenas, including: 1) cancer (non-small cell lung cancer, head and neck cancer, glioblastoma neoforme, ovarian cancer, and prostate cancer), 2) autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis, and myasthenia gravis), 3) pulmonary disease (idiopathic pulmonary fibrosis), 4) solid organ transplantation (lung, kidney, liver, and heart), and 5) inflammatory disorders.

Two of the areas that have been especially active over the past few years include the comprehensive immunologic profiling of cancer patients receiving so-called ‘immune checkpoint blockade’ therapies and the search for immune signatures in lung transplant recipients that track with resistance to CMV infection. The laboratory conducted immune monitoring studies associated with a Phase I trial of Ipilimumab (anti-CTLA-4) in a neoadjuvant setting for the treatment of non-small cell lung cancer (NSCLC). For this trial we extensively utilized several high parameter flow cytometry (PFC) platforms to follow activation, maturation, exhaustion, and proliferation patterns within CD4+ and CD8+ subsets of T-cells. We are also utilizing an intracellular cytokine staining (ICS) platform in efforts to detect anti-tumor associated antigen (TAA) responses by CD4+ and CD8+ T cells from peripheral blood mononuclear cells as well as lymphocytes infiltrating the patients’ tumor. These assays are designed to measure antigen-driven intracellular production of IFN-γ, TNF-α, and IL-2, as well as the degranulation marker CD107a. This strategy enables us to not only document individual cytokine responses, but to also assess (through Boolean gating) changes in relative polyfunctionality of the responses. We have also performed similar immune monitoring of a Phase II trial evaluating nivolumab (anti-PD-1) alone vs. combined nivolumamb + ipilimumab vs. avastin (bevacizamab) alone in patients with glioblastomas. In both studies, we are seeking to identify pharmacodynamics markers and immune correlates predictive of clinical responses. In completed studies of a cohort of lung transplant recipients, we identified specific polyfunctional signatures in CD4+ and CD8+ subsets against CMV pp65 and IE-1 antigens that tracked with resistance to CMV infection (manuscript in preparation). These findings now serve as the basis for a Phase I clinical trial to compare conventional 6-month chemoprophylaxis in lung transplant recipients versus a regimen dictated by the presence or absence of the predictive signatures. This trial is the principal component of a recently awarded Clinical Trials in Organ Transplantation or CTOT award made from the NIH to Duke (Scott Palmer, PI). Ongoing studies will test the hypothesis that these signatures that have been validated in lung transplant recipients will also predict resistance to CMV infection in the context of other solid organ transplants such as kidney, liver, and heart.Future studies will also attempt to identify predictive signatures for resistance to BK polyomavirus, the cause of graft threatening nephritis in kidney transplant recipients and cystitis in bone marrow transplant recipients.  

Recent publications

Zidar, D.A., Mudd, J.C., Juchnowski, S., Lopes, J.P., Sparks, S., Park, S.S., Ishikawa, M., Osborne, R., Washam, J.B., Chan, C., Funderburg, N.T., Owoyele, A., Alaiti, M.A., Mayuga, M., Orringer, C., Costa, M.A., Simon, D.I., Tatsuoka, C., Califf, R.M., Newby, L.K., Lederman, M.M., and Weinhold, K.J.  Altered maturation status and possible immune exhaustion of CD8 T lymphocytes in the peripheral blood of patients presenting with acute coronary syndromes. Arterioscler., Thromb., and Vasc. Biol. 36(2): 389-397, Feb. 2016 PMID: 26663396

Yi, J.S., Ready, N., Healy, P., Dumbauld, C., Berry, M., Shoemaker, D., Clarke, J., Crawford, J., Tong, B.C., Harpole, D., D’Amico, T.A., McSherry, F., Dunphy, F., McCall, S.J., Christensen, J.D., Wang, X, and Weinhold, K.J. Immune activation in early stage non-small cell lung cancer patients receiving neoadjuvant chemotherapy plus ipilimumab. Clin. Cancer Res. 23(24):7474-7482, 2017. PMCID: PMC5732888.

Reap, E., Suryadevera, C., Batuch, K., Sanchez-Perez, L., Archer, G., Schmittling, R., Norberg, P., Herndon II, J., Healy, P., Congdon, K., Gedeon, P., Campbell, O., Swartz, A., Riccione, K., Yi, J., Hossain-Ibrahim, M., Saraswathula, A., Nair, S., Anastasie, A., Broome, T., Weinhold, K.J., Desjardins, A., Vlahoviv, G., Mclendon, R., Firedman, H., Bigner, D., Fecci, P., Mitchell, D., and Sampson, J. Dendritic cells enhance polyfunctionality of adoptively transferred T cells which target cytomegalovirus in glioblastoma. Cancer Research 78(1):256-264, 2018. PMCID: PMC5754236.

Woroniecka, K., Chongsathidkiet, P., Rhodin, K., Kemeny, H., Dechant, C., Elsamadicy, A.A., Koyama, S., Jackson, C., Farber, H.S., Elsamadicy, A.A., Cui, X., Koyama, S., Jackson, C., Hansen, L., Bigner, D.D., Giles, A., Healy, P., Dranoff, G., Weinhold, K.J., Dunn, G.P., and Fecci, P.E. T cell exhaustion signatures vary with tumor type and are severe in glioblastoma. Clin. Cancer Res. Sep 1;24(17)4175-4186, 2018. PMCID: PMC6081269.

Weinhold, K.J., Bukowski, J.F., Brennan, T.V., Noveck, R.J., Staats, J.S., Lin, L., Stempora, L., Hammond, C., Wouters, A., Mojcik, C.F., Cheng, J., Collinge, M., Jesson, M.I., Hazra, A., Biswas, P., Lan, S., Clark, J.D., and Hodge, J.A. Reversibility of peripheral blood leukocyte phenotypic and functional changes after exposure to and withdrawal from tofacitinib, a Janus kinase inhibitor, in healthy volunteers. Clin. Immunology 191:10-20, June 19, 2018. PMCID: PMC6036921.

Berger, M., Oyeyemi, D, Olurinde, M.O., Whitson, H.E., Weinhold, K.J., Woldorff, M.G., Lipsitz, L.A., Moretti, E., Giattino, C.M., Rpberts, K.C., Zhou, J., Bunning, T., Ferrandino, M., Scheri, R.P., Cooter, M., Chan, C., Cabeza, R., Browndyke, J.N., Murdoch, D.M., Devinney, M.J., Shaw, L.M., Cohen, H.J., Mathew, J.P., and the INTUIT Investigators. The INTUIT Study: Investigating neuroinflammation underlying postoperative cognitive dysfunction. J. American Geriatrics Society 67940;794-798, 2019. PMCID: PMC6688749.

Berger, M., Murdoch, D., Staats, J., Chan, C., Thomas J., Garrigues, G., Browndyke, J., Cooter, M., Quinones, Q., Matthew, J., and Weinhold, K.J. Flow cytometry characterization of cerebrospinal fluid monocytes in patients with postoperative cognitive dysfunction (POCD): A pilot study. Anesthesia & Analgesia May 3, 2019 doi: 10.1213/ANE. PMCID: PMC6800758.

Nyanhete, T.E., Frisbee, A., Bradley, T., Faison, W.J., Robins, E., Payne, T.,Freel, S.A., Sawant, S., Weinhold, K.J., Wiehe, K., Haynes, B.F., Ferrari, G., Li, Q-J., Moody, M.A., and Tomaras, G.D. HLA class II-restricted CD8+T cells in HIV-1 virus controllers. Nat. Sci. Rep. 9(1):10165, 2019; PMCID: PMC6629643.

Yi, J.S., Rosa-Bray, M., Staats, J., Zakroysky, P., Chan, C., Russo, M., Dumbauld, C., White, S., Gierman, T., Weinhold, K.J., and Guptill, J.T. Establishment of normative ranges of the healthy immune system with comprehensive polychromatic flow cytometry profiling. PLoS One 14(12):e0225512, Dec.11, 2019. PMCID: PMC6905525.

Healy, Z.R., Weinhold, K.J., and Murdoch D.M. Transcriptional profiling of CD8+ CMV-specific T cell functional subsets obtained using a method for isolating high-quality RNA from fixed and permeabilized cells. Frontiers in Immunology 11:1859, Sep. 2, 2020. PMCID: PMC7492549.

Zhang, T., Harrison, M.R., O’Donnell, P.H., Ajjai, A., Hahn, N.M., Appleman, L.J., Cetnar, J., Burke, J.M., Fleming, M., Miloswsky. M., Mortazavi, A., Shore, N., Sonapavde, G., Schmidt, E., Bitman, B., Munugalavadla, V., Izumi, P., Patel, P., Staats, J., Chan, C., Weinhold, K.J.*and George, D.J.,*senior co-authors. A randomized phase 2 trial of pembrolizumab versus pembrolizumab and acalabrutinib in patients with platinum-resistant metastatic urothelial cancer. Cancer Oct.15, 2020 126(20):4485-4497. PMCID: PMC7590121

Salama, A.K.S., Palta, M., Rushing, C.N., Selim, M.A., Linnet, K.N., Czito, B.G., Yoo, D.S., Hanks, B.A., Beasley, G.M., Mosca, P., Dumbauld, C., Steadman, K.N., Yi, J.S., Weinhold, K.J., Tyler, D.S., Lee, W.T., and Brizel, D.M. Ipilimumab and radiation in patients with high risk resected or regionally advanced melanoma. Clin. Cancer Res. 1 March, 2021 27(5):1287-1295. PMCID: PMC8759408.

Li, Y., Yi, J.S., Russo, M.., Rosa-Bray, M., Weinhold. K.J., and Guptill, J.T. Normative dataset for plasma cytokines in healthy human adults. Data Brief 2021 Feb. 9;35:106857. PMCID: PMC7900339

White, S., Quinn, J., Enzor, E., Staats, J., Mosier, S.M., Almarode, J., Denny, T.N., Weinhold, K., Ferrari, G., and Chan, C. FlowKit: A Python toolkit for integrated manual and automated cytometry analysis workflows. Frontiers in Immunology 12:768541,Nov. 5, 2021. PMCID: PMC8602902.

Sung, B-Y., Lin, Y-H., Shah, P.D., Bieler, J.G., Palmer, S., Weinhold, K.J., Chang, H-R., Huang, H., Avery, R.K., Schneck, J., and Chiu, Y-L. Wnt activation promotes memory T cell polyfunctionality via epigenetic regulator PRMT1. J. Clin. Invest. 132(2):e140508, January 18, 2022. PMCID: PMC8759796.

Lusk, J.B., Quinones, Q.J., Staats, J.S., Weinhold, K.J., Grossi, P.M., Laskowitz, D.T., and James, M.L. Coupling hematoma evacuation with immune profiling for analysis of neuroinflammation after primary intracerebral hemorrhage: a pilot study. World Neurosurg. 2022 May;161:162-168 PMCID:PMID:35217228.

Brown, Landon C., Halabi, S., Somarelli, J., Humeniuk, M., Wu, Y., Oyekunle, T., Howard, L., Huang, J., Anand, M., Davies, C., Patel, P., Staats, J., Weinhold, K.J., Harrison, M.R., Zhang, T., George, D.J., and Armstrong, A.J. A phase 2 trial of avelumab in men with aggressive-variant or neuroendocrine prostate cancer. Prostate Cancer and Prostatic Diseases 25(4):762-769, 2022. PMCID: PMC8933335.

Khatri, A., Todd, J.L., Kelly, F.L., Nagler, A., Ji, Z., Jain, V., Gregory, S..G., Weinhold, K.J., and Palmer, S.M. JAK-STAT activation in basal cells contributes to cytotoxic T-cell mediated basal cell death in human chronic lung allograft dusfunction. JCI Insight 8(6) March 22, 2023 PMCID:PMC pending.

Zaffiri, L., Messinger, M., Staats, J.S., Patel, P., Palmer, S.M., Weinhold, K.J., Snyder, L.D., and Luftig, M.A. Evaluation of host cellular responses to Epstein-Barr virus (EBV) in adult lung transplant recipients with EBV-associated diseases. J. Med. Virol. 95(4):e28724, 2023.

The activities of the Ferrari Laboratory are based on both independent basic research and immune monitoring studies. The research revolves around three main areas of interest: class I-mediated cytotoxic CD8+ T cell responses, antibody-dependent cellular cytotoxicity (ADCC), gene expression in NK and T cellular subsets upon infection with HIV-1. With continuous funding over the last 11 years from the NIH and Bill & Melinda Gates Foundation along with many other productive collaborations within and outside of Duke, the Ferrari Lab has expanded its focus of research to include the ontogeny of HIV-1 specific immune responses that work by eliminating HIV-1 infected cells and how these can be induced by AIDS vaccine candidates.

Computational immunology (stochastic and spatial models and simulations, T cell signaling, immune regulation)
Statistical methodology for immunological laboratory techniques (flow cytometry, CFSE analysis, receptor-ligand binding and signaling kinetics)
Informatics of the immune system (reference and application ontologies, meta-programming, text mining and machine learning)