Duke Immune Profiling Core (DIPC)

• Tissue Processing and Biorepository
• Immune Phenotyping by High Dimension Polychromatic Flow Cytometry
• Multiplex Immunoassays
• Chip-Based Single Cell Proteomics Assays (Isolight)
• Bioinformatics and Biostatistics
• Computer Stations for Data Analysis

BD SORP Symphony A5
The LSRFortessa X50 high dimensional analyzer has a five-laser bench (405nm 200mW, 488nm 400mW, 532nm 1000mW, and 628nm 200mW, and 355nm 60mW) and is equipped with 44 fluorescence detectors, in addition to forward and side scatter detectors. Five decagons, coupled with the five higher-power lasers, tunable to variable power settings, allows for a total of 28 parameters to be measured simultaneously[JE1] [KWP2] . Laser Control Software allows for the adjustment of the individual laser powers as well as monitoring key performance parameters of the Coherent lasers. VXP electronics are configured for the detection of 46 parameters. The LSRFortessa X50 is equipped with a FACSFlow Supply System to regulate sample pressure accurately during high volume use, a microfluidics module that reduces coincidence and noise to enable acquisition of cell-limited samples, and a QuantiFlash LED-pulser to use for instrument performance qualification. The LSRFortessa X50 includes a reagent package to order from BD’s exclusive menu of high parameter Custom and Early Access reagents, High Parameter Technical Applications Support, and on-site training. Both cytometers are maintained on a service contract through the Shared Resource.
 

Luminex MAGPIX
The MAGPIX is a versatile multiplexing platform capable of performing qualitative and quantitative magnetic bead-based analysis of proteins and nucleic acids in a variety of sample matrices. This system can perform up to 50 tests in a single reaction volume, greatly reducing sample input, reagents and labor. The MAGPIX system features an innovative design based on LED excitation and CCD imaging technology that allows for a compact, robust system. MAGPIX is supported by a broad menu of commercially available assay kits, as well as reagents to build your own assays. Data Acquisition and Analysis for Luminex Instrumentation: xPONENT 4.1 is a modular, flexible, software package for control of the MAGPIX. The software’s graphical user interface follows the typical assay workflow but can be customized to meet the needs of a specific laboratory. For laboratories working under the FDA’s 21 CFR Part 11 regulations, xPONENT offers multilevel user management, full audit trail, electronic records and electronic signatures.

Isoplexis IsoLight Instrumentation
The Duke Immune Profiling Core (DIPC) is proud to announce the addition of an innovative new assay platform to serve the medical research enterprise across both the Duke Medical Center and Duke University. These chip-based, highly multiplexed and single cell proteomics platforms developed by Isoplexis Inc. are new to the Duke research community and have broad applicability across a wide variety of research disciplines such as novel cellular immunotherapies, solid organ transplantation, pre- and post-medical/surgical interventions, and general immunologic monitoring, to name but a few. The key component of this new platform is the IsoLight instrumentation (pictured below) that is now in place in DIPC.  Further details regarding the capabilities of the platform can be gleaned either from the Isoplexis website (isoplexis.com) or by contacting DIPC directly.  Please direct any questions to either Kent Weinhold (kjw@duke.edu) or Katelyn Steadman (katelyn.steadman@duke.edu).

Immediately following sample acquisition, all raw data (in the form of FCS files) are exported from the acquisition software to the local computer, then transferred to the local DHTS server in a single DIPC folder location, named Data. From there the data files are both downloaded to a local workstation for subsequent analysis and transferred to long-term storage for archival, with limited access. All investigators have access to download data files as needed from the DHTS Data folder; however, only Core staff and local IT have access to data in the archived folder, where the data are available as read only and may not be modified. All archived data are stored on the server permanently. When Core staff perform analysis for clinical trials, we maintain a folder for each trial; the folder includes two subfolders: Assay Docs (protocol or SOP, assay worksheet excel document, count sheets, etc) and Analysis Docs (FlowJo analysis file, a powerpoint summary of the analysis, excel containing data directly exported from FlowJo, completed assay and count sheets, and a record of any Deviations that occurred during the assay or analysis).

Server: File storage is managed and maintained by Duke Health Technology Services (DHTS).  It is an expandable NAS that is of enterprise grade, located in the Duke Fitz East Datacenter. It stores clinical and research information for other departments in the Duke Medical Center as well.  Called Isilon, this NAS grants us 30 days of retrievable copies of any file or folder.  As a result of this limitation and to achieve better data integrity, we have created a policy-based archive folder system that is separate but adjacent to the live data folders that our labs use to store data that will not be changed further. Data inside this archive is set as read-only for each group.

DIPC Leadership

• Kent J. Weinhold, PhD (Director)
• Cliburn Chan, PhD (Bioinformatics & Biostatistics)
• Janet S. Staats (Scientific/Research Manager)

DIPC Staff

• Jennifer Enzor
• Twan Weaver
• Preksha Patel
• Katelyn Steadman
• Erin Murray

Immune profiling for vaccine trials

• Supported by a multimillion dollar grant from NIH, DIPC will assess the cellular immune responses of volunteers enrolled in influenza and malaria vaccine trials through the Vaccine Trials Evaluation Unit (VTEU).
   

Immune profiling for cancer immunotherapy trials

1. Neoadjuvant ipilimumab therapy for non-small cell lung cancer (NSCLC); NCT01820754
2. Neoadjuvant Pembrolizumab therapy or non-small cell lung cancer; NCT02818920
3. Evaluation of nivolumab alone versus nivolumab + ipilimumab therapy for recurrent glioblastomas; NCT02017717
4. Treatment of urothelial cancers with pembrolizumab alone versus pembrolizumab + ACP196 (a BTK inhibitor); IND#124755
5. Evaluation of anti-tumor reactivities in the lymph node and PBMC of metastatic, castrate-resistant prostate cancer patients before or after receiving treatment with sipuleucel-T; NCT02036918NCT02036918
6. A randomized trial of Pembrolizumab and radiotherapy versus radiotherapy in high-risk soft tissue sarcoma of the extremity; NCT03092323

Representative DIPC Collaborative Publications

1. Maecker, H.T., McCoy, J.P. Jr., Amos, A., Elliott, J., Gaigalas, A., Wang, L., Aranda, R., Banchereau, J., Boshoff, C., Braun, J., Korin, Y., Reed, E., Cho, J., Hafler, D., Fathman, C.G., Robinson, W., Denny, T., Weinhold, K., Desai, B., Diamond, B., Gregersen, P., DiMeglio, P., Nestle, F., Peakman, M., Villnova, F., Ferbes, J., Field, E., Kantor, A., Kawabata, T., Komocsar, W., Lotze, M., Nepom, J., Ochs, H., O’Lone, R., Phippard, D., Plevy, S., Rich, S., Roederer, M.m Rostrosen, D., and Yeh, J.H.  A model for harmonizing flow cytometry in clinical trials.  Nat. Immunol. 11(11): 975-078, 2010. PMC3400260.
2. Snyder, L.D., Medinas, R., Chan, C., Sparks, S., Davis, W.A., Palmer, S.M., and Weinhold, K.J.  Polyfunctional cytomegalovirus-specific immunity in lung transplant recipients receiving valganciclovir.  Am. J. Transplant. 11:1-8, 2011. PMC3044779.
3. Murdoch, D.M., Staats, J.S., and Weinhold, K.J. OMIP-006: Phenotypic subset analysis of T regulatory cells via polychromatic flow cytometry.  Cytometry A 81(4):281-283, 2012. PMC3352600.
4. McNeil, L.K., Price, L., Britten, C.M., Jaimes, M., Maecker, H., Odunsi, K., Matsuzaki, J., Staats, J.S., Thorpe, J., Yuan, J., and Janetzki, S. International consortia proficiency panel conducted by the cancer immunotherapy consortium (CIC/CRI). Cytometry, Part A 83A:728-738, 2013. PMC4443815.
5. Richards, A.J., Staats, J., Enzor, J., McKinnon, K., Frelinger, J., Denny, T.N., Weinhold, K.J., and C. Chan. Setting objective thresholds for rare event detection in flow cytometry. J. Immunol. Methods, 409:54-61, 2014. PMC4138273.
6. Staats, J.S., Enzor, J.H., Sanchez, A.M., Roundtree, W., Guar, A., Jaimes, M., Denny, T.N., and Weinhold, K.J. Toward a comprehensive external quality assurance program for polychromatic flow cytometry. J. Immunol. Methods, 409:44-53, 2014. PMC4138230.
7. White, S., Laske, K., Welters, M.J.P., Bidmon, N., van der Burg, S.H., Britten, C.M., Enzor, J., Weinhold, K.J., Gouttefangeas, C., and Chan, C. Managing multi-center flow cytometry data for immune monitoring. Cancer Informatics 13(S7):111-122, 2014.  PMID:26085786
8. Snyder, L.D., Chan, C., Kwon, D., Yi, J.S., Martissa, J.A., Finlen Copeland, C.A., Osborne, R.J., Sparks, S.D., Palmer, S.M., and Weinhold, K.J.  Polyfunctional T cell signatures to predict protection from cytomegalovirus after lung transplant. Am. J. Respir. Crit. Care. Med. 193 (1): 78-85, Jan 1, 2016. PMC4731614.
9. 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. PMC5732888.
10. 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. PMC6036921.
11. 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. PMC6800758.
12. 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. PMC6905525.
13. 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. PMC7492549.
14. 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. Cancer Oct.15, 2020 126(20):4485-4497. PMC7590121.
15. 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. PMID:33172894.
16. 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. PMC7900339