Hematopoietic prostaglandin D synthase defines a proeosinophilic pathogenic effector human TH2 cell subpopulation with enhanced function Alyssa Mitson-Salazar,

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Presentation transcript:

Hematopoietic prostaglandin D synthase defines a proeosinophilic pathogenic effector human TH2 cell subpopulation with enhanced function Alyssa Mitson-Salazar, BS, Yuzhi Yin, MD, PhD, Daniel L. Wansley, PhD, Michael Young, RN, Hyejeong Bolan, RN, Sarah Arceo, RN, Nancy Ho, MD, Christopher Koh, MD, Joshua D. Milner, MD, Kelly D. Stone, MD, PhD, Stephen A. Wank, MD, Calman Prussin, MD Journal of Allergy and Clinical Immunology Volume 137, Issue 3, Pages 907-918.e9 (March 2016) DOI: 10.1016/j.jaci.2015.08.007 Copyright © 2015 Terms and Conditions

Fig 1 hPGDS and CD161 identify an IL-5bright subpopulation of TH2 cells. PBMCs from patients with EGID or NA subjects were activated with PMA and ionomycin and analyzed by means of flow cytometry. A, After gating on CD4+ T cells (columns 1-5) or CRTH2+ CD4 T cells (rightmost column), coexpression of TH2 cytokines with phenotype markers was analyzed. Data are representative of 7 patients with EGID and 7 NA subjects. Red arrows identify the analogous TH2 cell subpopulation in each dot plot. B, Expression of hPGDS by CD4+ TH2 cell subsets defined by CD161. C, The expression of CD161 by CD4+ TH2 cell subsets defined by hPGDS. In Fig 1, B and C, each symbol represents 1 patient with EGID. D, Boolean analysis of the frequency of CD4 T cells expressing combinations of the TH2 subset markers CRTH2, CD161, and hPGDS (see the key to the right of the bar graph). Each bar graph represents median values of 7 patients with EGID or 7 NA subjects. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig 2 hPGDS+CD161hi TH2 cells have greater TH2 cytokine expression. PBMCs from patients with EGID were activated as in Fig 1, and cytokine production among CD4 TH2 cell subpopulations was compared. A, After first gating on CRTH2+ CD4 T cells, TH2 cell subpopulations were identified according to the gating scheme in the rightmost column of Fig 1, A, and IL-5 versus IL-13 expression was analyzed. Data are representative of 7 subjects. B, Boolean analysis of TH2 cytokine expression in the above CRTH2+ subsets (n = 7 patients with EGID). C and D, Mean fluorescence intensity (MFI) of IL-5 (Fig 2, C) and IL-13 (Fig 2, D) in the above noted TH2 cell subsets. Each symbol represents 1 patient with EGID. E, TH2 cytokine expression of CRTH2+CD161hihPGDS+ T cells. Data are representative of 5 subjects. F, HDM-specific TH2 cell lines were activated and analyzed for hPGDS and CRTH2 expression. G, Boolean analysis of IL-5 and IL-13 expression within TH2 cell subpopulations of dust mite–specific TH2 cell lines. The key is the same as in Fig 2, B (n = 3 cell lines). H, Boolean analysis of cytokine coexpression within the noted CD4 T-cell subpopulations (n = 3 patients with EGID). Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig 3 hPGDS+ TH2 cell counts correlate with blood eosinophilia. Correlation of blood eosinophil counts with absolute numbers of blood TH2 cell subpopulations in adults with EGID (circles) and healthy nonatopic control subjects (diamonds; A and B) or patients with AD (C and D). Each symbol represents 1 patient. Spearman rank correlation was used. GI, Gastrointestinal. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig 4 peTH2 cells are highly differentiated effector memory T cells. A and B, Flow cytometric analysis of GATA3 (Fig 4, A) or eomesodermin (Eomes; Fig 4, B) expression in hPGDS+ TH2 cells (black line), hPGDS− TH2 cells (gray line), and non-TH2 CD4 T cells (light gray shading). Combined results of GATA3 and eomesodermin are shown to the right of the histograms (8 and 10 patients with EGID, respectively). MFI, Mean fluorescence intensity. C, Analysis of lymphocyte memory and differentiation markers of CD161hi TH2 cells (black line), CD161− TH2 cells (gray line), and non-TH2 CD4 T cells (light gray shading). Results are representative of 6 to 8 patients with EGID. D-F, Naive CD4 T cells underwent serial rounds of in vitro TH2 or TH1 differentiation and were analyzed for TH2 cytokine (Fig 4, D), hPGDS (Fig 4, E), and CD161 (Fig 4, F) expression. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig 5 peTH2 cells maintain a durable IL-5+ phenotype. A, Ex vivo CD4 T cells were analyzed for CD25 and HLA-DR after gating on CD161hi TH2 cells (black line), CD161− TH2 cells (gray line), and non-TH2 CD4 T cells (light gray shading). Data are representative of 8 patients with EGID. B, Parallel samples were analyzed for Ki-67 expression. C, Combined Ki-67 data from 8 patients with EGID. D, PBMCs from patients with EGID were labeled with Cell Trace Violet (CTV) and cultured in complete medium for 7 days, after which TH2 cell subpopulations were assessed for proliferation through dye dilution. E, Combined CTV proliferation data from 7 patients with EGID. F and G, Purified CD4 T cells were cultured for the indicated time in complete medium supplemented with 5 U/mL IL-2 (n = 3 cultures). Fig 5, F, shows TH2 cytokine production by hPGDS+ (black symbols) and hPGDS− (gray symbols) TH2 cells within these cultures after PMA/ionomycin activation. Fig 5, G, shows Boolean analysis of CD161 and hPGDS coexpression within the CRTH2+ TH2 cell population. In Fig 5, C, E, and F, each symbol represents 1 patient or culture. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig 6 peTH2 cells have an innate phenotype and function. A-E, IL-5 (Fig 6, A and B), IL-13 (Fig 6, C and D), and IL-9 (Fig 6, E) expression within the CD161− and CD161hi TH2 subsets was measured by using flow cytometry after activation with the indicated cytokine combinations (n = 3 patients with EGID). F, H, and I, Flow cytometric analysis of the indicated cytokine receptors in CD161hi TH2 cells (black line), CD161− TH2 cells (gray line), and non-TH2 CD4 T cells (light gray shading) in ex vivo PBMCs. Results are representative of 4 to 8 patients with EGID. G, Combined TSLP receptor (TSLP-R) data in TH2 cell subpopulations (n = 8 patients with EGID). J, Sorted CRTH2+CD161hi and CRTH2+CD161− CD4 T cells from 4 patients with EGID were activated with IL-2/IL-25/IL-33 and analyzed by means of quantitative PCR for expression of the indicated genes. Medium control and IL-2/IL-25/IL-33–stimulated TH2 cells are represented by gray and black bars, respectively. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig 7 peTH2 cells have an enhanced proallergic chemoattractant ligand and receptor program. A, Chemokine receptor expression was analyzed in CD161hi TH2 cells (black line), CD161− TH2 cells (gray line), and non-TH2 CD4 T cells (light gray shading). Data are representative of 6 to 9 patients with EGID. B, Combined CCR3 data from 6 patients with EGID. MFI, Mean fluorescence intensity. C, Sorted CD161− and CD161hi TH2 cells were analyzed for CCR3 by using quantitative PCR as in Fig 6, J. Gray bars, Media; black bars, IL-2/IL-25/IL-33 stimulated. D, Chemotaxis of CD161hi versus CD161− TH2 cells in response to eotaxin-1. Each symbol represents the cells from 1 patient. E, Sorted CD161hi and CD161− TH2 cells were activated, and supernatant PGD2 levels were measured. Unactivated and PMA/ionomycin-activated TH2 cells are represented by gray and black bars, respectively. Results are representative of 4 independent samples. F, Combined CXCR3 staining of TH2 subsets from 9 patients with EGID. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig 8 peTH2 cells express tissue-specific homing receptors and localize to sites of eosinophilic inflammation. A, Flow cytometric analysis of α4β7 and CLA expression by hPGDS+ TH2 cells (black line), hPGDS− TH2 cells (gray line), and non-TH2 CD4 T cells (light gray shading) from patients with EGID and those with AD. B and C, Combined analysis of α4β7 and CLA expression in hPGDS− TH2 2 (gray) and hPGDS+ TH2 (black) cells. Each symbol represents cells from 1 patient (n = 7 patients with EGID and 9 patients with AD). MFI, Mean fluorescence intensity. D, CRTH2 versus hPGDS expression in duodenal CD4 T cells from NA subjects and patients with EGID. E, Combined data showing the frequency of hPGDS+CD161hi TH2 cells in the antrum (squares), duodenum (circles), and esophagus (triangles) from NA subjects and patients with EGID. Each symbol represents 1 patient. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig E1 CD161 and hPGDS define a TH2 cell subpopulation. A, PBMCs from the indicated subject groups were activated and stained as in Fig 1 to examine IL-13, hPGDS, and CD161 coexpression by CD4 T cells. Data are representative of 7 patients with EGID and 7 NA subjects. The red arrows represent the analogous TH2 cell subpopulation in each dot plot. B, Correlation of the absolute peripheral blood cell count for the indicated TH2 cell subpopulations for patients with EGID (circles) and NA subjects (diamonds). Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig E2 Cytokine expression by TH2 cell subpopulations. A, PBMCs from patients with EGID were activated and stained to examine coexpression of IFN-γ and IL-13 in CRTH2− and CRTH2+ CD4 T-cell subpopulations. Data are representative of 7 subjects. B, Boolean analysis of the above showing combined results of 7 patients with EGID. C, Boolean analysis of IL-4/IL-5/IL-13 coexpression within the CRTH2+CD161hihPGDS+ CD4 T-cell subpopulation. The key is to the right of the pie chart. Each pie segment represents the median values of 5 patients with EGID. D, Respiratory syncytial virus (RSV)–specific TH1 and HDM-specific TH2 cell lines were analyzed for expression of CRTH2, hPGDS, and CD161 (n = 3-4 cell lines). E, IL-5 versus IL-9 dot plots in the noted TH2 cell subpopulations. Data are representative of 3 patients with EGID. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig E3 peTH2 cells are effector memory CD4 TH2 cells. A and B, CRTH2+CD161hi (black line), CRTH2+CD161− (gray line), and CRTH2−CD161− (light gray shading) T cells from PBMCs of patients with EGID were analyzed for expression of CD4 (Fig E3, A) and CD8 (Fig E3, B). C, CD161hi TH2 cells were examined for expression of the MAIT and natural killer T (NKT) cell invariant T-cell receptor (TCR) α chains Vα7.2 and Vα24. D, CD3 versus Lin-1 panel staining of CRTH2+CD161hi lymphocytes from PBMCs of patients with EGID. E, Combined data showing the relative percentage of peTH2 cells (CD3+Lin-1+) versus ILC2s (CD3−Lin-1−) within the CRTH2+CD161+ lymphocyte gate. F, Expression of hPGDS by ILC2s, defined as Lin−CD3−CRTH2+CD161hi lymphocytes in PBMCs of patients with EGID. Black line, Anti-hPGDS antibody; light gray shading, isotype control. G, Combined data showing hPGDS expression in ILC2s and a Lin−CD3−CRTH2−CD161− control lineage-negative cell population. H, PBMCs were activated with the noted stimuli, and cytokine production was measured after gating on either ILC2s or peTH2 cells. Data are representative of 5 patients with EGID. P/I, Phorbol myristate acetate, ionomycin. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig E4 Ex vivo phenotype of peTH2 cells. A-D, Combined results of lymphocyte memory (CD45RO, CCR7, CD62 L, and CD127) and differentiation (CD27 and programmed cell death 1 [PD-1]) markers (Fig E4, A), CD25 and HLA-DR (Fig E4, B), IL-17RB and ST2 (Fig E4, C), and CCR4 (Fig E4, D) in CD161hi and CD161− TH2 cells from donors with EGID. E, CCR8 (using clone 433H) expression by TH2 subpopulations after 24 hours of culture in medium or PHA (n = 6-8 different donors with EGID per experiment). MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions

Fig E5 Characterization of TH2 cell heterogeneity in patients with AD. A, CD161 expression by hPGDS+ and hPGDS− TH2 cells in patients with AD (n = 22). B, Flow cytometric analysis of TSLP receptor and CCR3 expression by hPGDS+ TH2 cells (black line), hPGDS− TH2 cells (gray line), and non-TH2 cells (light gray shading) in CD4 T cells from the indicated subject groups. GI, Gastrointestinal. C, Combined phenotype data of TH2 cell subsets from patients with AD. Each symbol represents 1 patient. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2016 137, 907-918.e9DOI: (10.1016/j.jaci.2015.08.007) Copyright © 2015 Terms and Conditions