Biology subjects

Pushpam, D.

Publications and source records attributed to Pushpam, D..

6 recordsLinked to original sources

bioRxiv2025

Dual Preconditioned Mesenchymal Stem Cells-Derived Culture Conditioned Media Augment Immunomodulation and Drive Metabolic Reprogramming in Acute Graft-versus-Host Disease

BackgroundThe toxicity associated with conventional conditioning regimens limits treatment outcomes in autoimmune disorders such as acute graft-versus-host disease (aGVHD). Developing effective adjunctive interventions to restore immune balance is therefore essential. This study investigated the immunomodulatory potential of mesenchymal stem cells (MSCs)-derived culture-conditioned media (CCM) from naive and preconditioned MSCs-hypoxic (MSCsHYP), apoptotic (MSCsAPO), and dual preconditioned (MSCsHYP+APO) in aGVHD. MethodsHuman MSCs isolated from bone marrow and Whartons Jelly were preconditioned under hypoxia (1% O2), apoptosis (1 {micro}M staurosporine, 24 h), or both. The immunoregulatory and antioxidant properties of their CCM were evaluated through T-cell proliferation assays, Treg induction, macrophage polarization, mitochondrial function, and T-cell bioenergetics. Comparative proteomic profiling of CCM from WJ-MSCs and WJ-MSCsHYP+APO co-cultured with aGVHD patient-derived activated PBMNCs was performed using LC-MS/MS, alongside in vivo validation in a chemotherapy-induced aGVHD murine model. ResultsWJ-MSCsHYP+APO-CCM exhibited superior immunomodulatory efficacy, suppressing T-cell proliferation, promoting Treg and Th2/Th9 differentiation, and driving M2 macrophage polarization. It reduced mitochondrial ROS, enhanced mitochondrial polarization, and shifted T-cell metabolism from glycolysis toward oxidative phosphorylation. Proteomic analysis revealed modulation of IL-12, IL-17, and JAK-STAT pathways, along with regulation of complement, coagulation, and metabolic cascades. Interaction with immune cells further enhanced its antioxidant and tissue-reparative properties via extracellular matrix remodeling. ConclusionDual preconditioning under hypoxia and apoptosis amplifies the immunomodulatory, antioxidant, and reparative efficacy of WJ-MSC-CCM, offering a potent non-cellular therapeutic strategy for aGVHD management. GRAPHICAL ABSTRACTImmunomodulatory and immune metabolic reprogramming potential of naive and preconditioned MSCs (MSCs, MSCsHYP, MSCsAPO, MSCsHYP+APO) derived CCM in Acute GVHD (created using Biorender.com) O_FIG O_LINKSMALLFIG WIDTH=95 HEIGHT=200 SRC="FIGDIR/small/686182v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@f7ed73org.highwire.dtl.DTLVardef@1063015org.highwire.dtl.DTLVardef@13ed827org.highwire.dtl.DTLVardef@e103c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

The HOTAIRM1-miR-222 Axis Regulates Venetoclax Resistance and Defines a High-Risk Subset in Pediatric t(8;21) Acute Myeloid Leukemia

Although acute myeloid leukemia (AML) with the RUNX1::RUNX1T1 fusion [t(8;21)(q22;q22.1)] defines a distinct cytogenetic subtype, differences in treatment response suggest additional molecular contributors beyond chromosomal abnormalities. Deregulated hematopoietic lineage-specific long non-coding RNAs (lncRNAs) contribute to leukemogenesis and therapy resistance. To investigate their role in t(8;21) AML, we performed whole-transcriptome sequencing of pediatric patients and age-matched healthy controls, identifying significant downregulation of lncRNA HOTAIRM1, a regulator of myeloid differentiation (adjusted P < 0.05). This was confirmed in a single-cell RNA-sequencing dataset (GSE116256) and the Leukemia MILE dataset (GSE13159, P=0.03). Validation of expression in our study cohort using qPCR specifically demonstrated significant downregulation of the myeloid specific isoform, HOTAIRM1 - HM1V2 (P<0.0001). Analysis of downstream pathways activated by HM1V2 loss identified miR-222, an oncomiR, as a de-repressed target (P=0.01). Elevated miR-222 expression was observed across AML cell lines (P<0.05), leukemic stem and progenitor cells (GSE117090, P<0.05), AML plasma-derived exosomes (GSE142699, P<0.0001), the current study dataset (P<0.0001), and the TARGET AML dataset (P<0.0001). Restoring HM1V2 expression with epigenetic agents azacytidine and panobinostat induced apoptosis in venetoclax-resistant Kasumi-1 cells (P < 0.01), through suppression of miR-222 (P < 0.01) and downregulation of anti-apoptotic proteins BCL-xL and MCL-1 (P < 0.05), key mediators of the venetoclax resistance mechanism. Machine learning based feature selection and Cox regression analysis showed that high miR-222 expression predicts poor outcome in pediatric t(8;21) AML, validated in both our institutional pediatric AML cohort (P < 0.05) and the multi-institutional TARGET cohort (P < 0.0001). Together, our findings highlight an epigenetic based approach to restore isoform-specific HM1V2 pathway function in venetoclax-resistant AML cells, and identifies miR-222 as a prognostic marker to refine risk stratification within the traditionally favorable-risk t(8;21) AML subgroup. Key PointsO_LILoss of myeloid lineage specific isoform of lncRNA HOTAIRM1 - HOTAIRM1 variant 2, results in de-repression of microRNA miR-222, and contributes to venetoclax resistance in pediatric AML patients harbouring the t(8;21)(q22;q22.1)/RUNX1::RUNX1T1 fusion. C_LIO_LIMicroRNA miR-222 shows potential as a single marker predictor that complements current risk stratification by identifying a subset of pediatric t(8;21) AML patients with poor prognosis. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/663834v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@19a0931org.highwire.dtl.DTLVardef@1d1a51aorg.highwire.dtl.DTLVardef@ae6783org.highwire.dtl.DTLVardef@de78e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Predictive Modeling of acute Graft-versus-Host-Disease using Machine Learning on Immune Cell and Cytokine Profiles at Engraftment

BackgroundAcute Graft-versus-Host-Disease (aGvHD) is a major immune complication following allogenic hematopoietic stem cell transplantation (Allo-HSCT), initiated by conditioning regimen-associated tissue damage. It involves the complex interplay of immune cells and cytokines. Our study aims to leverage machine learning (ML) algorithms on the immune and cytokine profile of Allo-HSCT recipients to develop biomarker-based classification models to predict the onset of aGvHD at the time of engraftment. Materials and MethodsSeventy patients diagnosed with hematological disorders who had undergone Ist Allo-HSCT were recruited from All India Institute of Medical Sciences, New Delhi, India. Peripheral blood (PB) was collected from the patients at the time of engraftment, and the immune cell subtypes and cytokine profiles were analyzed using flow cytometry and ELISA respectively. The individual cell counts were then processed using basic ML models, including support vector classifier with RBF kernel, Decision Tree, and Random Forest, chosen for their mathematical simplicity and feature importance advantage of Decision Trees and Random Forests. Various data settings were utilized in the study: combined immune and cytokine counts, immune cell counts only, cytokine counts only, T-cell counts only, NK cell counts only, dendritic cell counts only, and B-cell counts only. These configurations were selected to investigate how different data sets impact the prediction of aGvHD before its onset. ResultsAt the engraftment flow cytometric analysis of reconstituted lymphocytes in patients who developed aGvHD revealed that there was a remarkable decrease in the ratio of CD4+/CD8+ T-cell and Tregs, with an increase in the cytotoxic regulatory NK-cell, dendritic cells, and B-cell. The levels of pro-inflammatory cytokines (IFN-{gamma}, IL-1{beta}, IP-10, TNF-, IL-17, IL-12p70, MIP-1, MIP-1{beta}, RANTES), and Th17-and Th1-cells were elevated with consequent decline of the levels of anti-inflammatory cytokine IL-10, IL-2, IL-4 and Th2-, Th9-cells. Machine learning based on 48 parameters [all immune cell subsets n=34 and all cytokines (n=14)]. The correlation heat map shows a higher correlation of aGvHD with the cytokine profile with or without immune cells (accuracy: 1), T-cell alone (accuracy: 0.96); NK-cell alone (accuracy: 0.93); dendritic cells alone (accuracy: 0.90), B-cell alone (accuracy: 0.86). ConclusionThe current models classify perfectly, indicating the potential for a ML algorithm in predicting the onset of aGvHD. However, a study with a larger sample size is required to validate these classification models and mitigate the risk of overfitting observed due to the consistently high performance. The study also highlights the potential of cytokine profiles as a viable alternative to T-cell counts, as evidenced by the correlation heat map and classifier models. These findings provide valuable insights into dataset requirements and future directions for integrating ML models into aGvHD prediction.

immunology↗

bioRxiv2025

Dose-Response Immunomodulatory Effects of Mesenchymal Stem Cells-Derived Culture-Conditioned Media in Acute Graft-versus-Host-Disease

BackgroundMesenchymal stem cell-based therapy faces challenges that have driven interest in MSCs-derived culture-conditioned media (CCM) as a cell-free alternative. Our study aims to optimize the dose, and collection timing of CCM to enhance its therapeutic efficacy in aGVHD, while also standardizing co-culture conditions for CD3+ T-cell interaction with CCM. Material and MethodsHuman MSCs were isolated from BM and WJ and subsequently preconditioned under hypoxic conditions (1% O2) for 24 hours in a tri-gas incubator. Culture-conditioned media (CCM) was collected from both naive and hypoxia-preconditioned MSCs at 24, 48, and 72 hours and filtered using a 0.2 m membrane filter. CD3+ T-cell were isolated from PBMNCs derived from aGVHD patients. These T-cell were co-cultured at varying densities (2*106, 5*106, and 10*106 cells/ml) with different concentrations of CCM (25%, 50%, and 100%), and cell proliferation was assessed using the MTS assay. Furthermore, CD3+ T-cell proliferation and activation status were evaluated in a 2D co-culture model of CD3+ T-cell and CCM using flow cytometry. ResultsOur findings revealed that CCM collected at 48 hours, at a 50% concentration, exerted the most pronounced inhibitory effect on CD3+ T-cell proliferation, particularly at a density of 5*106 cells/ml, irrespective of the MSCs source. Hypoxia preconditioning significantly enhanced the immunomodulatory effects, with WJ-MSCsHYP-CCM demonstrating superior efficacy in suppressing T-cell proliferation, increasing the CD4+/CD8+ T-cell ratio, and reducing CD4+ T-cell activation compared to BM-MSCsHYP-CCM. ConclusionThese results emphasize the critical role of optimizing CCM collection timing and concentration to maximize therapeutic potential. Our study paves the way for the development of standardized, scalable, and effective cell-free therapies for aGVHD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/640019v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@16ecc48org.highwire.dtl.DTLVardef@12591dborg.highwire.dtl.DTLVardef@18af4beorg.highwire.dtl.DTLVardef@1c9e8c9_HPS_FORMAT_FIGEXP M_FIG C_FIG Optimum conditions for CCM dose, collection time point, and CD3+ T-cell dose for effective immunomodulatory effect of MSCs-derived CCM in aGVHD. (created using Biorender.com)

immunology↗

bioRxiv2025

Hypoxia-Mediated Molecular Interactions of Tissue-Specific Mesenchymal Stem Cells Drive Metabolic Reprogramming and Immunomodulation in Acute Graft-versus-Host Disease

BackgroundMesenchymal stem cells (MSCs) mediate immunomodulation through various mechanisms, including apoptosis, efferocytosis, and mitochondrial transfer. Our study investigates the impact of hypoxia preconditioning on the immune metabolic reprogramming and immunomodulatory potential of MSCs in acute graft-versus-host disease (aGVHD). Additionally, we explored the differential immunomodulatory effects of tissue-specific MSCs, specifically bone marrow (BM) and Whartons Jelly (WJ), and elucidated the mechanisms underlying variability in their therapeutic efficacy. MethodsMSCs were isolated from BM and WJ and subjected to hypoxia preconditioning. Their immunometabolic programming potential was assessed by evaluating T-cell proliferation, regulatory T-cell (Treg) induction, effector T-cell differentiation toward Th2, Th9 phenotypes, and macrophage polarization, T-cell bioenergetics in the direct co-culture systems. ResultsWJ-MSCsHYP exhibited superior immunomodulatory properties compared to BM-MSCsHYP, by inhibiting T-cell proliferation, enhancing Treg induction, and promoting anti-inflammatory macrophage polarization. WJ-MSCsHYP demonstrated enhanced mitochondrial transfer to T-cell, improving mitochondrial health, reducing ROS, and promoting oxidative phosphorylation, leading to immune homeostasis. Unlike BM-MSCs, WJ-MSCs exhibited higher rates of apoptosis, which facilitated immune modulation through mechanisms independent of efferocytosis. ConclusionOur findings highlight that WJ-MSCsHYP is a superior candidate for aGVHD by utilizing apoptosis, mitochondrial transfer, and metabolic reprogramming to achieve immune regulation.

immunology↗

bioRxiv2024

Synergistic Hypoxia and Apoptosis Conditioning Unleashes Superior Mesenchymal Stem Cells Efficacy in Acute Graft-versus-Host-Disease

Mesenchymal stem cells (MSCs) have emerged as promising candidates for immune modulation in various diseases that are associated with dysregulated immune responses like Graft-versus-Host-Disease (GVHD). MSCs are pleiotropic and the fate of MSCs following administration is a major determinant of their therapeutic efficacy. In this context, we here demonstrate that hypoxia preconditioned apoptotic MSCs [bone marrow (BM), Whartons Jelly (WJ)] bear more immune programming ability in a cellular model of acute Graft-versus-Host-Disease (aGVHD). To this purpose, we programmed MSCs by exposing them to hypoxia and inducing apoptosis both sequentially as well as simultaneously. Our findings demonstrated that WJ MSCs that were conditioned with indicated approaches simultaneously induced the differentiation of CD4+T-cell towards Tregs, enhanced Th2 effector, and concomitantly mitigated Th1 and Th17, with polarization of M1 effector macrophages towards their M2 phenotype, and more interestingly enhanced efferocytosis by macrophages indicated Th2 programming ability of MSCs programmed by conjunctional approaches Overall, our study highlights the potential of WJ-MSCs conditioned with hypoxia and apoptosis concurrently, as a promising therapeutic strategy for aGVHD and underscores the importance of considering MSC apoptosis in optimizing MSCs-based cellular therapy protocols for enhanced therapeutic efficacy in aGvHD. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC="FIGDIR/small/588248v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@92c102org.highwire.dtl.DTLVardef@bd895forg.highwire.dtl.DTLVardef@185e041org.highwire.dtl.DTLVardef@45ef0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗