Rapamycin preferentially inhibits human IL-5+ TH2-cell proliferation via an mTORC1/S6 kinase-1–dependent pathway Yuzhi Yin, MD, PhD, Alyssa Mitson-Salazar,

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Rapamycin preferentially inhibits human IL-5+ TH2-cell proliferation via an mTORC1/S6 kinase-1–dependent pathway Yuzhi Yin, MD, PhD, Alyssa Mitson-Salazar, BS, Daniel L. Wansley, PhD, Satya P. Singh, PhD, Calman Prussin, MD Journal of Allergy and Clinical Immunology Volume 139, Issue 5, Pages 1701-1704.e10 (May 2017) DOI: 10.1016/j.jaci.2016.10.029 Copyright © 2016 Terms and Conditions

Fig 1 Rapamycin preferentially inhibits allergen-driven TH2 cell output. A, Representative intracellular cytokine staining of CTV-stained CD4 T cells after a 7-day culture with HDM or RSV antigen. Rapamycin 1 nM was added as noted. B and C, Output cell count of CTVlow, cytokine+ CD4 subpopulations from HDM (TH2) and RSV (TH1) antigen-driven cultures after rapamycin and Torin1 dose response, n = 4 independent cultures, each from a different dust mite allergic subject (subjects 1-4). Fig 1, B and C, P < .0001 for the IL-5+ TH2 > IL-5− TH2 > TH1 trend, 2-way ANOVA. RSV, Respiratory syncytial virus. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig 2 peTH2 cells have greater S6K1 activity and rapamycin inhibition. A, S6RP phosphorylation by flow cytometry in phenotypically defined CD4 subpopulations. Combined data showing pS6RP in resting (B) and activated phenotypically defined CD4 subpopulations (C). D, Combined data showing inhibition of S6RP phosphorylation, 1 nM rapamycin vs vehicle. Fig 2, B-D, n = 4 independent experiments, each from a different subject (subjects 1-4). Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig E1 RSV and HDM, respectively, yield dominant TH1 and TH2 proliferative responses. Intracellular cytokine staining and CTV staining in CD4 T cells after 7-day culture with the indicated antigen. Results are representative of 4 independent cultures from donors 1 to 4. RSV, Respiratory syncytial virus. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig E2 Rapamycin preferentially inhibits in vitro differentiated TH2 cell proliferation. A and B, Fold change in output cell count in anti–CD3/CD28-activated 7-day cultures of in vitro differentiated TH1 and TH2 cells. C, The % inhibition due to 1 nM rapamycin was calculated from the above. D, ICCS profile of in vitro differentiated TH1 and TH2 cells after stimulation with anti-CD3/CD28 for 7 days, respectively, under TH1- or TH2-polarizing conditions. Fig E2, A-C, n = 3 independent experiments using cells from donors 2 to 4. D, Representative of same 3 experiments. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig E3 Rapamycin preferentially inhibits peTH2 cell proliferation. A-C, Phenotypic identification and validation of the intracellular cytokine staining profile of peTH2, cTH2, TH1, and naive CD4 T cells. PBMCs were activated with PMA/ionomycin for 6 hours, and stained for the noted phenotypic markers and cytokines. Cells were gated on CD3+, CD4+ cells and the phenotypic markers noted above the plot. D-H, Rapamycin inhibition of phenotypically defined effector T-cell subpopulations. PBMCs were stained with CTV, activated with anti-CD3, and cultured for 7 days. Fig E3, D and F, After first gating on CD4+ cells, the population of CTVlow cells is noted in the rectangular gate. E and G, After first gating on proliferated CTVlow cells, the frequencies of the above-noted phenotypic subpopulations are noted. H, Percent inhibition of output cell count of CTVlow phenotypically defined subpopulations (1 nM rapamycin vs vehicle). A-G, Results are representative of 4 independent cultures from donors 1 to 4. H, Combined data from donors 1 to 4. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig E4 Rapamycin does not preferentially induce apoptosis in TH2 cells. PBMCs were activated with soluble anti-CD3 (1 μg/mL) for 24 hours, during which they were treated with the inhibitors noted. A, Cleaved PARP staining in phenotypically defined CD4 subpopulations from a representative experiment. B, Grouped data of cleaved PARP staining in phenotypically defined CD4 subpopulations. n = 4 independent experiments, each from a different subject (subjects 1-4). PARP, Poly ADP-ribose polymerase. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig E5 Rapamycin does not inhibit 4E-BP1 phosphorylation. A, 4E-BP1 phosphorylation by flow cytometry, after first gating on each phenotypically defined CD4 subpopulation. Combined data showing p4E-BP1 in phenotypically defined CD4 subpopulations (B and C) and induction of 4E-BP1 phosphorylation by 1 nM rapamycin relative to vehicle (D). E, Western blotting for the noted mTOR effectors in cell extracts from in vitro differentiated TH2 cells. F, Combined data showing pmTOR in phenotypically defined CD4 subpopulations. G, Combined data showing pmTOR induction by 1 nM rapamycin relative to vehicle. Fig E5, B-D, F, and G. Results are representative of 5 independent cultures from donors 2 to 6. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig E6 Effect of S6K1 pathway knockdown on IL-5+ peTH2 cell proliferation. A, S6RP phosphorylation after siRNA knockdown of S6K1, S6RP, or both, in phenotypically defined CD4 subpopulations. Inhibition of proliferation of (B) phenotypically defined or (C) cytokine-defined CD4 subpopulations in S6K1/S6RP siRNA-treated PBMCs relative to scramble siRNA. Fig E6, A, Results are representative of 3 independent experiments. B and C, Results are representative of 6 independent cultures from donor 3 and donors 5 to 9. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions

Fig E7 Kinetics of pS6RP induction in human TH2 cells. In vitro differentiated TH2 cells were activated with plate-bound anti-CD3 (5 μg/mL) for the times indicated, or not activated (media) and then stained for pS6RP using phospho-flow according to the Methods section. Data are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2017 139, 1701-1704.e10DOI: (10.1016/j.jaci.2016.10.029) Copyright © 2016 Terms and Conditions