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Bet v 1–specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1–specific T-cell effector function in an activation-dependent.

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Presentation on theme: "Bet v 1–specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1–specific T-cell effector function in an activation-dependent."— Presentation transcript:

1 Bet v 1–specific T-cell receptor/forkhead box protein 3 transgenic T cells suppress Bet v 1–specific T-cell effector function in an activation-dependent manner  Klaus G. Schmetterer, MD, Daniela Haiderer, MSc, Victoria M. Leb-Reichl, PhD, Alina Neunkirchner, MSc, Beatrice Jahn-Schmid, PhD, Hans J. Küng, PhD, Karina Schuch, BSc, Peter Steinberger, PhD, Barbara Bohle, PhD, Winfried F. Pickl, MD  Journal of Allergy and Clinical Immunology  Volume 127, Issue 1, Pages e3 (January 2011) DOI: /j.jaci Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Scheme of multicistronic retroviral expression constructs used in this study. Function of the respective genes is indicated. Open boxes, genes; closed boxes, interposed picornaviral 2A or IRES elements. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Transgene expression levels and gating strategy for retrovirally transduced PB T cells. A, TRAV6+TRBV20+ T cells. B, TRAV6+TRBV20+FOXP3+, FOXP3+, and control T cells. In B, GFP expression served as additional gating marker. Surface TCR expression (TRBV20) and intracellular FOXP3 expression are displayed as 2 parameter dot plots. Numbers indicate cell percentages in respective quadrants. FSC, Forward scatter; SSC, side scatter. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Immunophenotype of transduced T cells. PB T cells gated for CD3+CD4+GFPneg or CD3+CD4+GFPhigh subsets and analyzed for expression of indicated Treg markers. Blue line, GFPhigh subset from TRAV6/TRBV20/FOXP3–transduced T cells, specific mAb; red line, GFPhigh subset from FOXP3-transduced T cells, specific mAb; green line, GFPneg subset, specific mAb; gray line, TRAV6/TRBV20/FOXP3–transduced T cells, control mAb. FSC, Forward scatter; SSC, side scatter. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 FOXP3-transduced T cells are hyporesponsive. Proliferation of retrovirally transduced PB T cells or nTreg cells stimulated with aAPCs expressing indicated molecules (A) or anti–CD3/CD28 coated microbeads in the absence (black bars) or presence (white bars) of recombinant human IL-2 (250 U/mL; B). Means + SDs of triplicates; control, empty IRES-GFP vector; asterisks indicate significance compared with TRAV6+TRBV20+ effector T cells (- IL-2). NS, Not significant. ∗∗∗P < kcpm, Kilo counts per minute. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 TRAV6/TRBV20/FOXP3–transduced T cells, unlike FOXP3-transduced T cells, suppress Bet v 1–specific effector T cells on allergen-specific activation. Proliferation of TRAV6+TRBV20+ transgenic effector T cells (Teff) cultured in the absence or presence of FOXP3+, TRAV6+TRBV20+FOXP3+ T cells, or nTreg cells at a 1:1 ratio on stimulation with Bet v 1–presenting aAPCs (A) or anti-CD3/CD28 coated microbeads (B). Data show means ± SDs from triplicates. Flow-cytometric analyses of eFluor 670 cell proliferation dye–labeled Teff in the absence (black line) or presence of FOXP3+ (red line), TRAV6+TRBV20+FOXP3+ T cells (blue line), or nTreg cells (green line) at a 1:1 ratio on stimulation with Bet v 1–presenting aAPCs (C) or anti-CD3/CD28 coated microbeads (D) in the absence (upper panel) or presence (lower panel) of exogenous IL-2 (250 U/mL). NS, Not significant. ∗∗∗P < kcpm, Kilo counts per minute. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Suppression of cytokine secretion of effector T cells by engineered Treg cells. A, Cytokine levels from cell culture supernatants collected after 24 hours (TNF-α, IL-2) or 48 hours (IFN-γ, IL-13). B, Intracellular staining of indicated cytokines. Numbers indicate percentages of cytokine positive cells among GFPneg (effector) cells. One representative experiment of several is shown. ∗P < .05; ∗∗P < .01; ∗∗∗P < CFSE, Carboxyfluorescein succinimidyl ester. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Percent suppression of titrated amounts of indicated transgenic Treg cells cocultured with effector T cells (Teff) on stimulation with Bet v 1–presenting aAPCs (A) or anti–CD3/CD28 coated microbeads (B). NS, Not significant. ∗P < .05; ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , e3DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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