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Proteomics and network analyses reveals inhibition of Akt-mTOR signaling in CD4+ T cells by Mycobacterium tuberculosis Mannose-capped Lipoarabinomannan

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, T Cell

SUBMITTER: Kathleen Lundberg

LAB HEAD: W. Henry Boom

PROVIDER: PXD004164 | Pride | 2018-07-05

REPOSITORIES: Pride

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Proteomics and Network Analyses Reveal Inhibition of Akt-mTOR Signaling in CD4+ T Cells by Mycobacterium tuberculosis Mannose-Capped Lipoarabinomannan.

Karim Ahmad F AF Sande Obondo J OJ Tomechko Sara E SE Ding Xuedong X Li Ming M Maxwell Sean S Ewing Rob M RM Harding Clifford V CV Rojas Roxana E RE Chance Mark R MR Boom W Henry WH

Proteomics 20171101 22

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose-capped lipoarabinomannan (ManLAM) inhibits CD4+ T-cell activation by inhibiting proximal T-cell receptor (TCR) signaling when activated by anti-CD3. To understand the impact of ManLAM on CD4+ T-cell function when both the TCR-CD3 complex and major costimulator CD28 are engaged, we performed label-free quantitative MS and network analysis. Mixed-effect model analysis of peptide intensity identified 149 unique pept ...[more]

PMID: 28994205

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Project description:Human regulatory T cells (TR) cells have potential for the treatment of immune mediated diseases, such as graft versus host disease, but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human TR cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9D4) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting TR cell proliferation, and under these conditions the proliferative capacity of TR cells was comparable to conventional CD4 lymphocytes. Blocking TGF-beta activity abrogated IL-10 expression from these cells, while addition of TGF-beta resulted in IL-10 production. These data demonstrate the ability of dendritic cells to provide proper costimulation to overcome the anergic phenotype of TR cells. In addition, these data demonstrate for the first time that TGF-beta is critical to enable TR cells to express IL-10. In order to confirm that our isolation strategy resulted in cells with characteristics of TR cells, we performed gene expression profiling of highly purified TR cells from two different individuals, which were compared to the gene expression profile of naÃ¯ve CD4+CD45RA+ cells. We first performed hierarchical clustering of the gene expression profiles of unactivated naÃ¯ve and TR cells from the two individuals. We show that the gene expression patterns of TR cells were highly similar between the two samples (correlation coefficient of 0.82), as were the gene expression profiles of naÃ¯ve CD4 cells between the two samples (correlation coefficient of 0.91). After averaging the gene expression signal from these two individuals, we compared the gene expression profile of unactivated TR cells with naÃ¯ve CD4 cells and show that TR cells over-express a variety of genes known to be associated with a TR phenotype (e.g. CTLA-4, IKZF2, IL-2RA). In addition, we also detected very high levels of expression of genes not typically associated with a TR phenotype, with the highest level of expression of IL-1 receptors in resting TR cells (Figure 2). These data confirm that the sorting strategy was effective at isolating T cells expressing a TR phenotype.

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	F045329.dat	Other
	F045329.mzid.gz	Mzid
	F045329.pride.mztab.gz	Mztab
	F045337.dat	Other
	F045337.mzid.gz	Mzid

Dataset Information

Proteomics and network analyses reveals inhibition of Akt-mTOR signaling in CD4+ T cells by Mycobacterium tuberculosis Mannose-capped Lipoarabinomannan

Dataset's files

Publications

Proteomics and Network Analyses Reveal Inhibition of Akt-mTOR Signaling in CD4<sup>+</sup> T Cells by Mycobacterium tuberculosis Mannose-Capped Lipoarabinomannan.

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