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Browsing by Subject "cytokine"

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  • Development of Cytokine Release Assay as a Prediction of Immunosafety 
    Liu, Jianyin (2022)
    Cytokine release syndrome is a severe systematic inflammatory disease that can be triggered upon pharmaceuticals intake. Evaluating the potential risk levels of novel therapeutics with an optimal assay is therefore essential. In this study, we tried to set up and validate a cytokine release assay from human peripheral blood mononuclear cells (PBMCs) for its application in nonclinical immunotoxicity assessments. Fresh PBMCs were isolated from buffy coats obtained from 11 healthy donors of different characteristics. Freshly isolated PBMCs were treated with LPS, positive control antibodies (anti-CD28, anti-CD3) and their corresponding isotypes (negative control antibodies) in both aqueous and solid formats to assess their abilities to induce cytokine release. Similarly, cryopreserved frozen PBMCs were also incubated with LPS, the positive control antibodies and the negative control antibodies, and compared their cytokine releasing capacities with freshly isolated PBMCs. A nine-cytokine panel (IFNγ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, TNFα, IL-12) was screened to select four cytokines (IFNγ, IL-2, IL-6, TNFα) in the following experimental setup. Freshly isolated PBMCs appeared to have higher sensitivity in response to the treatments as shown by the higher level of cytokine release. However, similar trends of cytokine release were observed between freshly isolated and frozen PBMCs in both aqueous and solid assay formats. LPS and anti-CD3 strongly induced cytokine release in all donors. Conversely, anti-CD28 induced cytokine release in some, but not all donors, possibly due to donor specificity. In summary, we have successfully developed and optimized a cytokine release assay, and it can be used to test the potential risk of immune-modulating drug candidate in the preclinical safety studies.
  • Vaccine- and infection-induced cellular immunity against SARS-CoV-2 in human 
    Vara, Saimi (2023)
    In this thesis, I studied T cell responses to SARS-CoV-2 structural proteins in subjects who had been both vaccinated and infected (n=30), who had only been infected (n=22), and as controls, in subjects who had been neither vaccinated nor infected (n=6). In addition, I compared cellular responses between groups of subjects who had been infected with either wild-type (WT) SARS-CoV-2, Alpha (B.1.1.7), or Beta (B.1.351) variants. Before analyzing the samples to be studied, I optimized the conditions for the cell stimulations. Peripheral blood mononuclear cells (PBMCs) were collected from infected subjects six months after infection. PBMCs were stimulated with SARS-CoV-2 wild-type nucleoprotein, spike-, envelope-, and membrane protein peptide pools. I quantified cytokines and effector molecules characteristic of CD4+ and CD8+ cell responses; perforin, tumor necrosis factor alpha (TNF-a), granzyme B, interferon gamma (IFN-γ), interleukin 2 (IL-2) and interleukin 4 (IL-4) secreted by PBMCs were quantified. In this study, I found that subjects with infection, or combination of infection and vaccination had higher cellular immune responses compared to uninfected controls. Infection induced higher granzyme B, IFN-y, and IL-2 secretion, and the combination of infection and vaccination induced higher granzyme B, perforin, IFN-y, IL-2 and IL-4 secretion. I found that subjects with hybrid immunity, defined as immunity acquired from combined vaccination and infection, had on average higher IL-4 responses compared to those who had been infected only. In this study, I found that nucleoprotein, spike-, and membrane proteins stimulated T cell responses whereas envelope protein did not stimulate T cell responses. I found that WT, Alpha or Beta infection produced equally good T cell responses to WT spike peptide. In conclusion, I found that COVID-19 patients have long-lasting T cell responses. I found that T cells recognize different SARS-CoV-2 variants. Mutations present in the spike proteins of the different variants do not affect T-cell ability to recognize these antigens. Immunity based on T cells is not as susceptible to antigenic changes as the humoral immunity. T cells have a vital role in protection against variants, when new SARS-CoV-2 variants evaded antibody-based immunity.

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