Understanding the alterations in the cytokine profiles of Lymphocytes under variable activation conditions

Majumder, Sudipta (2025) Understanding the alterations in the cytokine profiles of Lymphocytes under variable activation conditions. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Sudipta Majumder (20MS138)) 20MS138_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

The tumor microenvironment is characterized by altered ionic conditions, notably elevated extracellular potassium ([K⁺]ₑₓₜ), which can profoundly influence immune cell function. This study investigates the impact of high [K⁺]ₑₓₜ on lymphocyte signaling, focusing on both T and B cells. Jurkat E6.1 (T cells) and Ramos-RA1 (B cells) were treated with increasing concentrations of [K⁺]ₑₓₜ, followed by its activation. While Ramos-RA1 cells exhibited minimal changes in total phosphorylation and Syk activation levels under elevated potassium conditions, Jurkat T cells showed significant alterations in phosphorylation patterns and cytokine expression profiles, indicating a cell type-specific sensitivity to ionic stress. Further analysis using ZAP-70-deficient Jurkat P116 cells, revealed that the modulation of cytokine responses by [K⁺]ₑₓₜ is dependent on ZAP-70 signaling. To dissect the mechanistic role of ZAP-70, a kinase-dead mutant (HRD→HRN) was generated via site-directed mutagenesis and validated through sequencing and immunoblotting. A Syk-mCherry construct was similarly prepared in a lentiviral backbone for comparative analysis. Additionally, a mouse model was used to mimic the tumor-like ionic microenvironment, where localized autoimmune-like inflammation was observed in the joints, though liver histology remained unaffected. Our findings underscore a differential regulation of lymphocyte signaling by extracellular potassium, with T cells being more responsive than B cells. The study lays the groundwork for future investigations into immune cell behavior under ionic stress and highlights the potential of targeting ionic homeostasis in modulating immune responses within the tumor microenvironment.

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