bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428853; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Quantitative analysis and genome-scale modeling of human CD4+ T-cell differentiation reveals subset-specific regulation of pathways

Running title: Metabolic modeling of human CD4+ T-cells on a genome-scale

Partho Sen1,2,*, Syed Bilal Ahmad Andrabi1, Tanja Buchacher1, Mohd Moin Khan1, Ubaid Ullah1, Tuomas Lindeman1, Marina Alves Amaral1, Victoria Hinkkanen1, Esko Kemppainen1, Alex M Dickens1,3, Omid Rasool1, Tuulia Hyötyläinen4, Riitta Lahesmaa1,*, Matej Orešič1,2,*

1Turku Bioscience Centre, University of Turku and Åbo Akademi University, 20520 Turku, Finland 2School of Medical Sciences, Örebro University, 702 81 Örebro, Sweden 3Department of Chemistry, University of Turku, 20520 Turku, Finland 4Department of Chemistry, Örebro University, 702 81 Örebro, Sweden

*Correspondence: Partho Sen, PhD. Phone: +358 46 960 8145; Email: [email protected] Riitta Lahesmaa, M.D., PhD. Email: [email protected] Matej Orešič, PhD. Email: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428853; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

ABSTRACT T-cells are sentinels of adaptive cell-mediated immune responses. T-cell activation, proliferation and differentiation involves metabolic reprogramming involving the interplay of , and metabolites. Here, we aim to understand the metabolic pathways involved in the activation and functional differentiation of human CD4+ T-cell subsets (Th1, Th2, Th17 and iTregs). We combined genome-scale metabolic modeling, expression data, targeted and non-targeted lipidomics experiments, together with in vitro gene knockdown experiments and showed that human CD4+ T- cells undergo specific metabolic changes during activation and functional differentiation. In addition, we identified and confirmed the importance of and glycosphingolipid synthesis pathways in Th17 differentiation and effector functions. Finally, through in vitro gene knockdown experiments, we substantiated the requirement of palmitoyl (SPT), a de novo pathway in the expression of proinflammatory cytokine (IL17A and IL17F) by Th17 cells. Our findings may provide a comprehensive resource for identifying CD4+ T-cell-specific targets for their selective manipulation under disease conditions, particularly, diseases characterized by an imbalance of Treg / Th17 cells. Our data also suggest a role for elevated levels of in conditions comorbid with these diseases, e.g., obesity and insulin resistance.

KEYWORDS: CD4+ T-cells; Ceramides; Gene expression; Genome-scale metabolic modeling; Glycosphingolipid ; ; Lipidomics; Metabolic pathways; .

2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.29.428853; this version posted January 31, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

INTRODUCTION CD4+ T-cells play a central role in the adaptive immune system. They orchestrate the immune responses and mediate prote