Dataset Information

Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways

ABSTRACT: Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell-receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon co-stimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in presence of PRBCs. Inhibitory activity of PRBCs required direct cell-cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species (ROS), which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in presence of PRBCs. Phosphorylation of the TCR-related zeta-chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. Only downstream signaling events requiring ROS for full functionality were affected, confirmed by an untargeted mass spectrometry-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than did treatment with 1 mM of the anti-oxidant N-acetyl cysteine. Taken together, our data suggest that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects might be responsible for clinical observations associated with transfusion of PRBCs.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): T Cell, T-lymphocyte

SUBMITTER: Christopher Gerner

LAB HEAD: Christopher Gerner

PROVIDER: PXD023144 | Pride | 2021-06-01

REPOSITORIES: Pride

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Packed red blood cells inhibit T-cell activation via ROS-dependent signaling pathways.

Gerner Marlene C MC Bileck Andrea A Janker Lukas L Ziegler Liesa S LS Öhlinger Thomas T Raeven Pierre P Müllner Ernst W EW Salzer Ulrich U Gerner Christopher C Schmetterer Klaus G KG Baron David M DM

The Journal of biological chemistry 20210101

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimu ...[more]

PMID: 33676898

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Project description:CD4+ T cells have a central role in controlling Mtb infection (1). Despite immune control, Mtb persists by interfering with macrophage and T cell function, allowing for pathogen survival. Our group has demonstrated direct and indirect inhibition of CD4+ T cell activation by different Mtb molecules including lipoproteins LpqH, LprA and LprG, and ManLAM (2-5). ManLAM is an abundant glycolipid in the Mtb cell wall and interferes with TCR signaling by down-regulating phosphorylation of Lck, CD3, ZAP70 and LAT (6). Activation through the TCR-CD3 complex leads to marked changes in the proteome of T cells. Entry into cell cycle, production of cytokines and differentiation from naïve to effector and memory T cell are all reflected in the production and modification of multiple proteins. Mass spectrometry (MS) has been used to characterize TCR complex formation (7-11) and the effect of a range of stressors on the T cell proteome (12-14). Recent advances have overcome technical issues in quantitative MS and allow analysis of the complexity and dynamic range of the cellular proteome (15). To extract biological meaning different bioinformatic tools have been developed for MS based cellular studies (16, 17). In this study we used label-free quantitative MS to characterize the effect of ManLAM on the proteome of activated CD4+ T cells. Approximately 5000 peptides were identified and quantitated from three biological experimental datasets in primary murine CD4+ T cells treated with or without ManLAM and activated with anti-CD3 and anti-CD28 monoclonal antibodies (mAb). Peptides with varying abundance were selected by likelihood ratio based statistical significance by comparing the intensities under different experimental conditions. ManLAM treatment resulted in abundance changes for 149 peptides representing 131 proteins in the activated CD4+ T cell proteome that affected a variety of enriched cluster modules and pathways. Validation experiments confirmed Man LAM induced inhibition of Proliferating Cell Nuclear Antigen (PCNA) that regulates cell cycle progression. Protein-protein interaction (PPI) network analysis revealed a central role for the Akt/mTOR signaling pathway. WB studies revealed decreased phosphorylation of both Akt and mTOR in ManLAM treated CD4+ T cells as well as decreased expression of Otub1, a key molecule regulated by mTOR. Thus disruption of the activated CD4+ T cell proteome by ManLAM-mediated inhibition of proximal TCR signaling is primarily mediated through inhibition of Akt-mTOR signaling.

			Action	DRS
	20-Phos-1361.tdf_bin	Other
	20-Phos-1362.tdf_bin	Other
	20-Phos-1363.tdf_bin	Other
	20-Phos-1364.tdf_bin	Other
	20-Phos-1365.tdf_bin	Other