Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells.

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2020-01

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Abstract

Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.

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10.3389/fimmu.2020.01859

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Healy, Zachary R, Kent J Weinhold and David M Murdoch (2020). Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells. Frontiers in immunology, 11. p. 1859. 10.3389/fimmu.2020.01859 Retrieved from https://hdl.handle.net/10161/21776.

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Scholars@Duke

Weinhold

Kent James Weinhold

Joseph W. and Dorothy W. Beard Distinguished Professor of Experimental Surgery

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


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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.

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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.

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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

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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.

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Murdoch

David Martin Murdoch

Associate Professor of Medicine

As a physician and researcher, my career has been driven by a passion for linking the basic and clinical sciences with the primary goal of understanding the disease pathogenesis. Through my training in epidemiology, basic science immunology, and clinical medicine, I have acquired a breadth of experience, knowledge, collaborators, and an adaptability which has culminated in a research focus on the reconstitution of immune responses and systemic inflammation in immunocompromised patients and vulnerable populations. My research focuses on T cell immunology utilizing a variety of platforms including polychromatic flow cytometry, cytokine multiplexing, and novel single cell assays. My initial research centered on the immune reconstitution syndrome (IRIS), with a focus on the mycobacterial precipitants of the disease, its epidemiology, and research efforts into elucidating the pathogenesis of the syndrome. Recently, I have translated my interest in co-infection immunology in the immunocompromised transplant population. With a career long interest in contrasting compartmental and peripheral immune responses, I have partnered with engineers in the Duke Pratt School of Engineering in order to develop novel single cell immune assays in order to comprehensively profile the immune response on limited specimens.


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