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bioRxiv · 10.1101/2024.09.05.611044

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

Mendiratta, M.·Rai, S.·Gupta, R.·Bakhshi, S.·Aggarwal, M.·Gupta, A. K.·Prakash, H.·Mohanty, S.·Sahoo, R. K.
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Abstract

BackgroundT-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 patients 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 (1-10g/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 (5-10g/ml), whereas aGVHD patients exhibited heightened proliferation even at lower PHA concentrations (1-2.5g/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 ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/611044v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@ab3c47org.highwire.dtl.DTLVardef@2786forg.highwire.dtl.DTLVardef@30c698org.highwire.dtl.DTLVardef@3e4821_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights of the studyO_LILower doses of PHA (1.0g/ml) and IL-2 (50IU/ml) are optimum conditions for aGVHD patients derived CD3+ T-cell proliferation under in vitro conditions. C_LIO_LIThe maximum T-cell proliferation in healthy individuals occurs with 7.5g/ml PHA and 50IU/ml IL-2. C_LIO_LIHigher doses of PHA induce cytotoxicity in both cohorts. C_LIO_LIIL-2 significantly enhances T-cell survival, with 50IU/ml maintaining robust proliferation over extended periods. C_LI

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Mendiratta, M., Rai, S., Gupta, R., Bakhshi, S., Aggarwal, M., Gupta, A. K., Prakash, H., Mohanty, S., Sahoo, R. K.. 2024-09-09. A Comparative assessment of T-Cell response of Healthy donors and Acute Graft-versus-Host-Disease Patients: Customizing immune monitoring platform. https://doi.org/10.1101/2024.09.05.611044

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