A Comparative assessment of T-Cell response of Healthy donors and Acute Graft-versus-Host-Disease Patients: Customizing immune monitoring platform

Abstract

AbstractBackgroundT-cell activation and proliferation are critical for understanding immune responses in both healthy and pathological conditions such as acute graft-versus-host disease (aGVHD). Phytohemagglutinin (PHA) and interleukin-2 (IL-2) are commonly used in in vitro assays to study T-cell responses. In view of the discrete response of T cells from aGvHD patient’s cohorts, our study optimized PHA / IL-2 based T-cell response among healthy individuals versus aGVHD patients.MethodsPeripheral blood was collected from age- and sex-matched healthy individuals (n=10) and aGVHD patients (n=10). CD3+T-cell were isolated and stimulated with varying concentrations of PHA (1-10μg/ml) and IL-2 (50-500 IU/ml). Cell proliferation was assessed using MTS and CFSE assays, while their apoptosis was evaluated with Annexin V/7-AAD staining.ResultsWe observed enhanced proliferation of healthy individuals at higher PHA concentrations (5-10μg/ml), whereas aGVHD patients exhibited heightened proliferation even at lower PHA concentrations (1-2.5μg/ml) at 48 hours. Prolonged exposure of T cells from GvHD patients to PHA led to decreased proliferation while it increased in the T cells from healthy donors.IL-2 supplementation (50 IU/ml) of T-cells from healthy donors significantly enhanced their proliferation and survival, with the optimal concentration supporting robust proliferation over extended culture periods.ConclusionOur study optimized PHA and IL-2 concentrations required for T-cell proliferation studies among healthy individuals and aGVHD patients. and underscored experimental conditions required for studying T-cell behavior/dysregulation in aGVHD condition.GRAPHICAL ABSTRACTHighlights of the studyLower doses of PHA (1.0μg/ml) and IL-2 (50IU/ml) are optimum conditions for aGVHD patients derived CD3+ T-cell proliferation underin vitroconditions.The maximum T-cell proliferation in healthy individuals occurs with 7.5μg/ml PHA and 50IU/ml IL-2.Higher doses of PHA induce cytotoxicity in both cohorts.IL-2 significantly enhances T-cell survival, with 50IU/ml maintaining robust proliferation over extended periods.