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Metabolic control of regulatory T cell (Treg) survival and function by Lkb1

  1. Ronald M. Evansa,d,1
  1. aGene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037;
  2. bStorr Liver Centre, Westmead Institute for Medical Research and Sydney Medical School, Westmead Hospital, University of Sydney, Westmead, NSW 2145, Australia;
  3. cNomis Laboratories for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, CA 92037;
  4. dHoward Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037
  1. Contributed by Ronald M. Evans, October 5, 2017 (sent for review August 31, 2017; reviewed by Chih-Hao Lee and Ming O. Li)

  1. Fig. 2.

    Ablation of Lkb1 abrogates Treg function and results in uncontrolled immune activation. (A) Flow cytometry analysis of CD44 and CD62L expression on CD4+ T cells in spleen and peripheral LNs of WT and KO mice (n = 3–5). (B) Flow cytometry analysis of cytokine production by splenic CD4+ T cells from WT and KO mice (n = 3–5). (C) Treg percentage in spleen and LNs of 3- to 4-wk-old WT and KO mice (n = 3–5). (D) In vitro suppression of CFSE-labeled WT na?ve CD4+ T responder cells (Tresp) by WT and KO Tregs. (EG) WT or KO Tregs purified from CD45.1?CD45.2+ mice and Teffs (CD4+CD45RBhiCD25?) isolated from CD45.1+CD45.2? mice were mixed and transferred in to Rag1?/? recipient mice (n = 5) through retroorbital injection. Changes in body weight (F) and H&E staining of colon sections (magnification: 40×) (G) of each group are shown. Data are shown as mean ± SD and are representative of three experiments. Statistical significance was determined by Student's unpaired t test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

  2. Fig. 3.

    Lkb1 is required for Tregs to maintain cellular metabolism and energy homeostasis. (A) Mitochondrial mass and membrane potential of freshly purified WT and KO Tregs assayed by Mitotracker and DilC1(5) staining, respectively. (B) OCR and ECAR in freshly purified WT and KO Tregs under basal conditions and in response to sequential addition of oligomycin, fluorocarbonyl cyanide phenylhydrazone (FCCP), rotenone (Rtn), and antimycin A (AA). (C and D) ATP concentrations and ROS levels in freshly purified WT and KO Tregs, respectively. All data are representative of three experiments and are expressed as mean ± SD. (E and F) KEGG pathway analysis and functional annotation of down-regulated gene categories in KO Tregs, respectively. (G) Representative down-regulated gene lists in KO Tregs. Statistical significance was determined by Student's unpaired t test (**P < 0.01; ***P < 0.001).

  3. Fig. 4.

    Lkb1 deletion compromises Treg survival, and its downstream kinases MARKS and SIKS mediate Lkb1 function in Tregs. (A) Flow cytometry analysis of survival of WT and KO Tregs over time in vitro. (B) WT or KO Tregs purified from CD45.1?CD45.2+ mice and Teffs (CD4+CD45RBhiCD25?) isolated from CD45.1+CD45.2? mice were mixed and retroorbitally injected in Rag1?/? recipient mice (n = 5). Cellularity was analyzed at 5 wk after initial transfer. (C) Apoptosis in WT and KO Tregs assayed by cleaved caspase-3 staining after in vitro culturing for 12 h. (D) DNA damage response in WT and KO Tregs assayed by phosphor-H2A.x staining after in vitro culturing for 12 h. (E) Expression profile of Lkb1 downstream kinases in Tregs. (F) Flow cytometry analysis of the survival of WT Tregs in response to different combination of inhibitors targeting AMPK, MARKS, SIKS, and NUAKS. All data are representative of three experiments. Statistical significance was determined by Student's unpaired t test [not significant (n.s.): P > 0.05; ***P < 0.001; ****P < 0.0001].

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