Characterisation and analysis of Memory T-cells in COVID-19 patient sample
Ho, Hai Wen
Ho, Hai Wen
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Publication Date
2023-08-08
Type
Thesis
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Abstract
Background: Immunological memory is the key to long-term protection against disease. It is the hallmark of adaptive immunity which facilitates an accelerated and enhanced immune response against a pathogen encountered previously, providing better clearance than naïve T cells response. Memory subsets of T-cell includes central memory T-cell (TCM), effector memory T-cell (TEM), terminally differentiated effector memory T-cell (TEMRA). Defining these memory cells against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) is therefore crucial in understanding the long-term protection against it. Longitudinal analysis of patient infected with SARS-CoV2 have demonstrated persistent virus-specific memory T-cells up to 10 months post-infection, but a lot remains to be learned. Aims: The aims of this thesis were (1) to understand the difference in T-cell and its’ memory subsets in individuals who had different severity of COVID-19, (2) to evaluate the T-cell memory response upon stimulation with SARS-CoV2 Spike protein, and 3) to determine the cytokine response upon stimulation and its correlation with the memory T-cell profiles. Methods: Severity of COVID-19 was defined based on WHO clinical progression scale where mild disease was defined as asymptomatic or symptomatic but doesn’t require hospitalization; moderate disease as requiring hospitalization but not requiring non-invasive ventilation (NIV); severe disease as hospitalized requiring NIV, invasive ventilation, or mechanical ventilation. Blood samples were collected from COVID-19 patients and Peripheral Blood Mononuclear Cells (PBMCs) were isolated via centrifugation with a Ficoll density gradient. Isolated PBMCs were stimulated with SARS-CoV2 Spike Protein Cocktail of wild type, alpha, beta, and delta strains and incubated for 24 hours at 37°C, 5% CO2. Cells were characterised by flow cytometry and supernatants were analysed for cytokine concentration by enzyme-linked immunosorbent assay (ELISA). Blood samples from the Irish Blood Transfusion Service (IBTS) were collected and used as healthy control. Parametric data were analysed with ordinary one-way ANOVA with Tukey’s multiple comparison tests while non-parametric data were analysed using Kruskal-Wallis tests with Dunn’s multiple comparison test. Correlation of T cell memory subsets before and after stimulation with the concentration of cytokines was analysed using Pearson rank order correlation Strength of the correlation were determined based on the correlation coefficient. Results: At baseline, COVID-19 donor PBMCs HLA-DR mean fluorescence index (MFI) of CD4+ T-cell, 1322 (297.17) were significantly elevated in the severe group compared to healthy (p= 0.0026) and mild (p=0.0272) disease groups. HLA-DR MFI of CD8+ T-cells also showed a similar trend of increasing MFI with increasing disease severity although not statistically significant. CD4+ memory T-cell subsets showed a significant reduction of CD4+ TEMRA frequency in severe disease, 4.69%, compared to healthy donors 29.53% (p= 0.024). CD4+ TEM trended down from healthy donor to severe disease group whereas CD4+ TCM trended in the opposite direction in the severe disease group although neither were not statistically significant. In CD8+ memory T-cell subsets, no significant differences have been identified between different disease severity groups although the frequency of CD8+ TEM and CD8+ TCM shows an increase with increasing disease severity. Upon stimulation with SARS CoV2 Spike protein, the greatest expansion of CD4+ and CD8+ TEM frequencies were noted in severe disease groups with the ratio of stimulated to unstimulated measuring at 1.207 (CD4+ TEM) and 1.227 (CD8+ TEM). An increase in HLA-DR MFI on CD4+ has been identified in moderate and severe COVID-19 groups with the MFI ratio of stimulated to unstimulated of 1.119 and 1.032 but were not statistically significant. Concentrations of cytokines (IL-6, TNF-α, IFN-γ ) were noted to increase with disease severity. At baseline (unstimulated) significant correlation has been identified between CD4+ T-cells HLA-DR MFI and IFN-γ concentration, r =0.7596, p=0.0067; CD4+ TEMRA and IL-6 concentration, r =0.7282, p=0.0110; CD4+ TEMRA and TNF-α concentration, r =0.7045, p=0.0155. Upon stimulation of PBMCs with SARS-CoV2 Spike Protein, significant correlation has been identified between the change in CD4+ T-cell HLA-DR MFI and TNF-α concentration, r =-0.6029, p=0.0496; CD4+ T-cell HLA-DR MFI and IFN-γ concentration, r =0.7515, p=0.0076; CD4+ TEM and IFN-γ concentration, r =0.6773, p=0.0220; CD8+ Naïve T-cell and IFN-γ concentration, r =0.6996, p=0.0165. Conclusion: Individuals who had a more severe initial COVID-19 infection, at baseline, T cells showed a higher level of activation which further increases on stimulation indicating a hyperactivation state of T-cells possibly contributing to disease severity. Their T-cell memory profile showed a lower proportion of effector memory subsets. However, upon stimulation, greatest expansion of effector memory subset, TEM along with higher production of cytokines IL-6, TNF-a, and IFN-y which correlate with the T-cell profile changes. This suggests a greater immune recall response in individuals who had severe initial infection.
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NUI Galway