Skip to main content
SpringerLink
Log in

Human T Cell Priming Assay: Depletion of Peripheral Blood Lymphocytes in CD25+ Cells Improves the In Vitro Detection of Weak Allergen-Specific T Cells

  • Chapter
  • First Online:
T Lymphocytes as Tools in Diagnostics and Immunotoxicology

Part of the book series: Experientia Supplementum ((EXS,volume 104))

Abstract

To develop an in vitro assay that recapitulates the key event of allergic contact dermatitis (ACD), that is the priming of effector T cells by hapten-presenting dendritic cells, and then allows for the sensitive detection of chemical allergens represents a major challenge. Classical human T cell priming assays (hTCPA) that have been developed in the past, using hapten-loaded monocyte-derived dendritic cells (MDDCs) as antigen-presenting cells and peripheral blood lymphocytes (PBLs) as responding cells, were not efficient to prime T cells to common allergens with moderate/weak sensitizing properties. Recent progress in the understanding of the effector and regulatory mechanisms of ACD have shown that T cell priming requires efficient uptake of allergens by immunogenic DCs and that it is controlled by several subsets of regulatory cells including CD25+ Tregs. We therefore analyzed various parameters involved in allergen-specific T cell activation in vitro and showed that priming of allergen-specific T cells is hampered by several subsets of immune cells comprising CD1aneg DCs, CD25+ T cells, and CD56+ regulatory cells.

CD4+CD25+FoxP3+ Tregs prevented the in vitro T cell priming to moderate/weak allergens, and depletion of human PBLs in CD25+ cells significantly increased specific T cell proliferation and IFN-γ secretion. CD56+ cells exerted an additional control of T cell priming since co-depletion of both CD56+ and CD25+ cells improved the magnitude of chemical-specific T cell activation. Finally, CD1alow MDDCs were able to inhibit T cell activation obtained by allergen-pulsed CD1ahigh MDDC. Moreover, we showed that uptake by DC of allergen-encapsulated nanoparticles significantly increased their activation status and their ability to prompt specific T cell activation. Hence, by combining the different strategies, i.e., depletion of CD25+ and CD56+ cells, use of CD1ahigh MDDC, and nanoparticle encapsulation of allergens, it was possible to induce T cell priming to most of the moderate/weak allergens, including lipophilic molecules highly insoluble in culture media. Therefore, the present optimized in vitro human T cell priming assay is a valuable method to detect the sensitizing properties of chemical allergens.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

ACD:

Allergic contact dermatitis

DCs:

Dendritic cells

DMSO:

Dimethyl sulfoxide

DNCB:

2,4-Dinitrochlorobenzene

FITC:

Fluorescein isothiocyanate

HCA:

a-Hexylcinnamaldehyde

hTCPA:

human T cell priming assay

ISs:

Immunological synapses

mAb:

monoclonal antibody

MDDCs:

Monocyte-derived dendritic cells

MDLR:

Mixed dendritic cell lymphocyte reaction

PBLs:

Peripheral blood lymphocytes

PBMCs:

Peripheral blood mononuclear cells

SLS:

Sodium lauryl sulfate

TNBS:

2,4,6-Trinitrobenzene sulfonic acid

References

  • Akiba H, Kehren J, Ducluzeau MT et al (2002) Skin inflammation during contact hypersensitivity is mediated by early recruitment of CD8+ T cytotoxic 1 cells inducing keratinocyte apoptosis. J Immunol 168(6):3079–87

    PubMed  CAS  Google Scholar 

  • Bursch LS, Wang L, Igyarto B et al (2007) Identification of a novel population of Langerin+dendritic cells. J Exp Med 204(13):3147–56

    Article  PubMed  CAS  Google Scholar 

  • Cohen MH, Chretien PB, Felix EL et al (1974) Augmentation of lymphocyte reactivity in guinea pigs, mice, monkeys and humans sensitised to BCG, dinitrochlorobenzene or nitrogen mustard. Nature 249(458):656–8

    Article  PubMed  CAS  Google Scholar 

  • Dustin ML (2010) Insights into function of the immunological synapse from studies with supported planar bilayers. Curr Top Microbiol Immunol 340:1–24

    Article  PubMed  CAS  Google Scholar 

  • Feau S, Garcia Z, Arens R, Yagita H, Borst J, Schoenberger SP (2012) The CD4(+) T-cell help signal is transmitted from APC to CD8(+) T-cells via CD27-CD70 interactions. Nat Commun 3:948

    Article  PubMed  Google Scholar 

  • Gomez DE, Aguero M, Vocanson M, Hacini-Rachinel F et al (2012) Langerhans cells protect from allergic contact dermatitis in mice by tolerizing CD8(+) T cells and activating Foxp3(+) regulatory T cells. J Clin Invest 122(5):1700–11

    Article  Google Scholar 

  • Gorbachev AV, Fairchild RL (2010) CD4+CD25+ regulatory T cells utilize FasL as a mechanism to restrict DC priming functions in cutaneous immune responses. Eur J Immunol 40(7):2006–15

    Article  PubMed  CAS  Google Scholar 

  • Goubier A, Vocanson M, Macari C et al (2013) Invariant NKT cells suppress CD8(+) T-cell-mediated allergic contact dermatitis independently of regulatory CD4(+) T cells. J Invest Dermatol 133(4):980–7

    Article  PubMed  CAS  Google Scholar 

  • Henrickson SE, Mempel TR, Mazo IB et al (2008) In vivo imaging of T cell priming. Sci Signal 1(12); pt2

    Google Scholar 

  • Honda T, Egawa G, Grabbe S, Kabashima K (2013) Update of immune events in the murine contact hypersensitivity model: toward the understanding of allergic contact dermatitis. J Invest Dermatol 133(2):303–15

    Article  PubMed  CAS  Google Scholar 

  • Iyori M, Zhang T, Pantel H, Gagne BA, Sentman CL (2010) TRAIL/DR5 plays a critical role in NK cell-mediated negative regulation of dendritic cell cross-priming of T cells. J Immunol 187(6):3087–95

    Article  Google Scholar 

  • Kaplan D, Igyarto BZ, Gaspari AA (2012) Early immune events in the induction of allergic contact dermatitis. Nat Rev Immunol 12(2):114–24

    PubMed  CAS  Google Scholar 

  • Kehren J, Desvignes C, Krasteva M et al (1999) Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity. J Exp Med 189(5):779–86

    Article  PubMed  CAS  Google Scholar 

  • Krasteva M, Peguet-Navarro J, Moulon C, Courtellemont P, Redziniak G, Schmitt D (1996) In vitro primary sensitization of hapten-specific T cells by cultured human epidermal Langerhans cells–a screening predictive assay for contact sensitizers. Clin Exp Allergy 26(5):563–70

    Article  PubMed  CAS  Google Scholar 

  • Lass C, Vocanson M, Wagner S et al (2008) Anti-inflammatory and immune-regulatory mechanisms prevent contact hypersensitivity to Arnica montana L. Exp Dermatol 17(10):849–57

    Article  PubMed  CAS  Google Scholar 

  • Le Borgne M, Etchart N, Goubier A et al (2006) Dendritic cells rapidly recruited into epithelial tissues via CCR6/CCL20 are responsible for CD8+ T cell crosspriming in vivo. Immunity 24(2):191–201

    Article  PubMed  Google Scholar 

  • Macleod AS, Havran WL (2011) Functions of skin-resident gamma delta T cells. Cell Mol Life Sci 68(14):2399–408

    Article  PubMed  CAS  Google Scholar 

  • Mahnke K, Ring S, Johnson TS et al (2007) Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation. Eur J Immunol 37(8):2117–26

    Article  PubMed  CAS  Google Scholar 

  • Martin S, Lappin MB, Kohler J et al (2000) Peptide immunization indicates that CD8+ T cells are the dominant effector cells in trinitrophenyl-specific contact hypersensitivity. J Invest Dermatol 115(2):260–6

    Article  PubMed  CAS  Google Scholar 

  • Martin S, Delattre V, Leicht C, Weltzien HU, Simon JC (2003) A high frequency of allergen-specific CD8+ Tc1 cells is associated with the murine immune response to the contact sensitizer trinitrophenyl. Exp Dermatol 12(1):78–85

    Article  PubMed  CAS  Google Scholar 

  • Martin SF, Dudda JC, Bachtanian E et al (2008) Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity. J Exp Med 205(9):2151–62

    Article  PubMed  CAS  Google Scholar 

  • Martin SF, Esser PR, Schmucker S (2010) T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays. Cell Mol Life Sci 67(24):4171–84

    Article  PubMed  CAS  Google Scholar 

  • Martin SF, Esser PR, Weber FC et al (2011) Mechanisms of chemical-induced innate immunity in allergic contact dermatitis. Allergy 66(9):1152–63

    Article  PubMed  CAS  Google Scholar 

  • Mattarollo SR, Yong M, Gosmann C (2011) NKT cells inhibit antigen-specific effector CD8 T cell induction to skin viral proteins. J Immunol 187(4):1601–8

    Article  PubMed  CAS  Google Scholar 

  • Moed H, Von Blomberg M, Bruynzeel DP, Scheper R, Gibbs S, Rustemeyer T (2005) Improved detection of allergen-specific T-cell responses in allergic contact dermatitis through the addition of ‘cytokine cocktails’. Exp Dermatol 14(8):634–40

    Article  PubMed  CAS  Google Scholar 

  • Nakashima H, Hamaguchi Y, Watanabe R et al (2010) CD22 expression mediates the regulatory functions of peritoneal B-1a cells during the remission phase of contact hypersensitivity reactions. J Immunol 184(9):4637–45

    Article  PubMed  CAS  Google Scholar 

  • Ohkura N, Sakaguchi S (2010) Regulatory T cells: roles of T cell receptor for their development and function. Semin Immunopathol 32(2):95–106

    Article  PubMed  CAS  Google Scholar 

  • Pandiyan P, Zheng L, Ishihara S, Reed J, Lenardo MJ (2007) CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells. Nat Immunol 8(12):1353–62

    Article  PubMed  CAS  Google Scholar 

  • Peiser M, Tralau T, Heidler J et al (2012) Allergic contact dermatitis: epidemiology, molecular mechanisms, in vitro methods and regulatory aspects: Current knowledge assembled at an international workshop at BfR, Germany. Cell Mol Life Sci 69:763–81

    Article  PubMed  CAS  Google Scholar 

  • Richter A, Schmucker SS, Esser PR et al (2013) Human T cell priming assay (hTCPA) for the identification of contact allergens based on naive T cells and DC—IFN-gamma and TNF-alpha readout. Toxicol In Vitro 27:1180–5

    Article  PubMed  CAS  Google Scholar 

  • Ring S, Schafer SC, Mahnke K, Lehr HA, Enk AH (2006) CD4+ CD25+ regulatory T cells suppress contact hypersensitivity reactions by blocking influx of effector T cells into inflamed tissue. Eur J Immunol 36(11):2981–92

    Article  PubMed  CAS  Google Scholar 

  • Ring S, Karakhanova S, Johnson T, Enk AH, Mahnke K (2010) Gap junctions between regulatory T cells and dendritic cells prevent sensitization of CD8(+) T cells. J Allergy Clin Immunol 125(1):237–46, e231–237

    Article  PubMed  CAS  Google Scholar 

  • Rougier N, Redziniak G, Mougin D, Schmitt D, Vincent C (2000) In vitro evaluation of the sensitization potential of weak contact allergens using langerhans-like dendritic cells and autologous T cells. Toxicology 145(1):73–82

    Article  PubMed  CAS  Google Scholar 

  • Rozieres A, Vocanson M, Rodet K et al (2010) CD8+ T cells mediate skin allergy to amoxicillin in a mouse model. Allergy 65(8):996–1003

    Article  PubMed  CAS  Google Scholar 

  • Rustemeyer T, De Ligter S, Von Blomberg BM, Frosch PJ, Scheper RJ (1999) Human T lymphocyte priming in vitro by haptenated autologous dendritic cells. Clin Exp Immunol 117(2):209–16

    Article  PubMed  CAS  Google Scholar 

  • Schildknecht A, Brauer S, Brenner C et al (2010) FoxP3+ regulatory T cells essentially contribute to peripheral CD8+ T-cell tolerance induced by steady-state dendritic cells. Proc Natl Acad Sci U S A 107(1):199–203

    Article  PubMed  CAS  Google Scholar 

  • Semmling V, Lukacs-Kornek V, Thaiss CA et al (2010) Alternative cross-priming through CCL17-CCR4-mediated attraction of CTLs toward NKT cell-licensed DCs. Nat Immunol 11(4):313–20

    Article  PubMed  CAS  Google Scholar 

  • Shevach EM (2011) Biological functions of regulatory T cells. Adv Immunol 112:137–76

    Article  PubMed  Google Scholar 

  • Soderquest K, Walzer T, Zafirova B et al (2011) Cutting edge: CD8+ T cell priming in the absence of NK cells leads to enhanced memory responses. J Immunol 186(6):3304–8

    Article  PubMed  CAS  Google Scholar 

  • Speiser DE, Baumgaertner P, Voelter V et al (2008) Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen. Proc Natl Acad Sci U S A 105(10):3849–54

    Article  PubMed  CAS  Google Scholar 

  • Tang Q, Bluestone JA (2008) The Foxp3+ regulatory T cell: a jack of all trades, master of regulation. Nat Immunol 9(3):239–44

    Article  PubMed  CAS  Google Scholar 

  • Tang Q, Krummel MF (2006) Imaging the function of regulatory T cells in vivo. Curr Opin Immunol 18(4):496–502

    Article  PubMed  CAS  Google Scholar 

  • Vocanson M, Goujon C, Chabeau G et al (2006a) The skin allergenic properties of chemicals may depend on contaminants–evidence from studies on coumarin. Int Arch Allergy Immunol 140(3):231–8

    Article  PubMed  CAS  Google Scholar 

  • Vocanson M, Hennino A, Cluzel-Tailhardat M et al (2006b) CD8+ T cells are effector cells of contact dermatitis to common skin allergens in mice. J Invest Dermatol 126(4):815–20

    Article  PubMed  CAS  Google Scholar 

  • Vocanson M, Cluzel-Tailhardat M, Poyet G et al (2008) Depletion of human peripheral blood lymphocytes in CD25+ cells allows for the sensitive in vitro screening of contact allergens. J Invest Dermatol 128:2119–22

    Article  PubMed  CAS  Google Scholar 

  • Vocanson M, Hennino A, Rozieres A, Poyet G, Nicolas JF (2009) Effector and regulatory mechanisms in allergic contact dermatitis. Allergy 64(12):1699–714

    Article  PubMed  CAS  Google Scholar 

  • Vocanson M, Rozieres A, Hennino A et al (2010) Inducible costimulator (ICOS) is a marker for highly suppressive antigen-specific T cells sharing features of TH17/TH1 and regulatory T cells. J Allergy Clin Immunol 126(2):280–9, 289 e281–287

    Article  PubMed  CAS  Google Scholar 

  • Wing JB, Sakaguchi S (2012) Multiple treg suppressive modules and their adaptability. Front Immunol 3:178

    Article  PubMed  CAS  Google Scholar 

  • Yamaguchi T, Wing JB, Sakaguchi S (2011) Two modes of immune suppression by Foxp3(+) regulatory T cells under inflammatory or non-inflammatory conditions. Semin Immunol 23(6):424–30

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. CIRI - International Center for Infectiology Research, Inserm, U1111, Université Lyon 1, 21 Avenue Tony Garnier, 69007, Lyon, France

    Marc Vocanson, Amine Achachi, Virginie Mutez, Aurore Rozières, Philippe Fournier & Jean-François Nicolas

  2. Allergology & Clinical Immunology, CH Lyon-Sud, Pierre-Benite, France

    Marc Vocanson, Amine Achachi, Virginie Mutez, Aurore Rozières, Philippe Fournier & Jean-François Nicolas

  3. LVMH Recherche, LVMH Parfums et Cosmétiques, St Jean de Braye, 45804, France

    Magalie Cluzel-Tailhardat

  4. Links, Montelier, 26120, France

    Béatrice Le Varlet

Authors
  1. Marc Vocanson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amine Achachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Virginie Mutez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Magalie Cluzel-Tailhardat
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Béatrice Le Varlet
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Aurore Rozières
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Philippe Fournier
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean-François Nicolas
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marc Vocanson .

Editor information

Editors and Affiliations

  1. Department of Dermatology and Venereology, University Medical Center Freiburg, Freiburg, Germany

    Stefan F. Martin

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Basel

About this chapter

Cite this chapter

Vocanson, M. et al. (2014). Human T Cell Priming Assay: Depletion of Peripheral Blood Lymphocytes in CD25+ Cells Improves the In Vitro Detection of Weak Allergen-Specific T Cells. In: Martin, S. (eds) T Lymphocytes as Tools in Diagnostics and Immunotoxicology. Experientia Supplementum, vol 104. Springer, Basel. https://doi.org/10.1007/978-3-0348-0726-5_7

Download citation

Publish with us

Policies and ethics

Search

Navigation