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

Background: T-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 dynamics. Our study aimed to determine the optimal concentrations of PHA and IL-2 for promoting T-cell proliferation and survival and to evaluate how these conditions impact T-cell responses in healthy individuals versus aGVHD patients. Methods: Peripheral blood samples were 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 apoptosis was evaluated with Annexin V/7-AAD staining. Results: We observed enhanced proliferation of healthy individuals at higher PHA concentrations (5-10μg/ml), whereas aGVHD patients exhibited heightened proliferation at lower PHA concentrations (1-2.5μg/ml) at 48 hours. Prolonged exposure to PHA led to decreased proliferation in aGVHD patients, while healthy individuals continued to show increased proliferation at 72 hours with optimal PHA concentrations of 5.0-7.5μg/ml. The CFSE assay confirmed these findings, showing a higher proliferation rate in healthy individuals at elevated PHA concentrations and in aGVHD patients at lower concentrations. IL-2 supplementation (50 IU/ml) significantly enhanced T-cell proliferation and survival, with the optimal concentration supporting robust proliferation over extended culture periods. Conclusion: Our study identifies optimal PHA and IL-2 concentrations for in vitro T-cell studies, with 7.5μg/ml of PHA and 50IU/ml of IL-2 providing robust T-cell proliferation in healthy individuals. In contrast, aGVHD patients' T-cells showed better proliferation at lower PHA concentrations (1.0μg/ml) and similar IL-2 requirements. These results underscore the need for tailored experimental conditions based on patient profiles to effectively study T-cell behavior and improve therapeutic strategies for T-cell dysregulation in aGVHD and other conditions.