{"title":"A Comparative assessment of T-Cell response of Healthy donors and Acute Graft-versus-Host-Disease Patients: Customizing immune monitoring platform","authors":"Mohini Mendiratta, Meenakshi Mendiratta, Sandeep Rai, Ritu Gupta, Sameer Bakhshi, Mukul Aggarwal, Aditya Kumar Gupta, Hridayesh Prakash, Sujata Mohanty, Ranjit K Sahoo","doi":"10.1101/2024.09.05.611044","DOIUrl":null,"url":null,"abstract":"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.\nMethods: 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.\nConclusion: 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.","PeriodicalId":501182,"journal":{"name":"bioRxiv - Immunology","volume":"7 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"bioRxiv - Immunology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1101/2024.09.05.611044","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
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.