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Mapping of the autophagosomal degradome identifies IL-7Rα as key 1 cargo in proliferating CD4+ T-cells

ABSTRACT: CD4+ T cells orchestrate both humoral and cytotoxic immune responses. While it is known that CD4+ T cell proliferation relies on autophagy, direct identification of the autophagosomal cargo involved is still missing. Here, we created a transgenic mouse model, which, for the first time, enables us to directly map the proteinaceous content of autophagosomes in any primary cell by LC3 proximity labelling. IL-7Rα, a cytokine receptor mostly found in naïve and memory T cells, was reproducibly detected in autophagosomes of activated CD4+ T cells. Consistently, CD4+ T cells lacking autophagy showed increased IL-7Rα surface expression, while no defect in internalisation was observed. Mechanistically, excessive surface IL-7Rα sequestrates the common gamma chain, impairing the IL-2R assembly and downstream signalling crucial for T cell proliferation. This study provides proof-of-principle that key autophagy substrates can be reliably identified with this model to help mechanistically unravel autophagy’s contribution to healthy physiology and disease.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cytotoxic T-lymphocyte Protein 4 (mouse), Mef Cell

SUBMITTER: Roman Fischer

LAB HEAD: Roman Fischer

PROVIDER: PXD033733 | Pride | 2022-07-08

REPOSITORIES: pride

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			Action	DRS
	FL1317_MSQ2103_20210129_DZhou_1_Ctrl1.raw	Raw
	FL1317_MSQ2103_20210129_DZhou_2_ctrl2.raw	Raw
	FL1317_MSQ2103_20210129_DZhou_3_ctrl3.raw	Raw
	FL1317_MSQ2103_20210129_DZhou_4_RAPI1.raw	Raw
	FL1317_MSQ2103_20210129_DZhou_5_RAPI2.raw	Raw

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Project description:Autophagy has been implicated as a host defense mechanism against intracellular pathogens. However, certain intracellular pathogens such as Leishmania can manipulate the host’s autophagy to promote their survival. Recently, our findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of mammalian target of rapamycin complex 1 regulation. This occurs in the background of enhanced mTOR activity, which suggests the fine-tuning of autophagy to optimally promote parasite survival and may involve the sequestration or modulation of specific autophagosome-associated proteins. To investigate how Leishmania potentially manipulates the composition of host-cell autophagosomes, we undertook a quantitative proteomic study of the human monocytic cell line THP-1 following infection with L. donovani. We used stable isotope labeling by amino acid in cell culture and liquid chromatography-tandem mass spectrometry to compare expression profiles between autophagosomes isolated from THP-1 cells infected with L. donovani or treated with known autophagy inducers. Select proteomics results were validated by immunoblotting. In this study, we showed that L. donovani modulates the composition of macrophage autophagosomes during infection when compared to autophagosomes induced by either rapamycin (selective autophagy) or starvation (non-selective autophagy). Among 1787 proteins detected in Leishmania-induced autophagosomes, 146 were significantly modulated compared to the proteome of rapamycin-induced autophagosomes while 57 were significantly modulated compared to starvation-induced autophagosomes. Strikingly, 23 Leishmania proteins were also detected in the proteome of Leishmania-induced autophagosomes. Together, our data provide the first comprehensive insight into the proteome dynamics of host autophagosomes in response to Leishmania infection and demonstrate the complex relations between the host and pathogen at the molecular level.

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Mapping of the autophagosomal degradome identifies IL-7Rα as key 1 cargo in proliferating CD4+ T-cells

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