BACKGROUND: Major histocompatibility complex (MHC) class II tetramers (tetramers) allow to detect allergen-specific CD4(+) T cells at a single-cell level. Limits to this technology include HLA restriction and the need to identify immunodominant T cell epitopes. OBJECTIVE: Assessing the expression of various activation markers following allergen stimulation to replace tetramer staining. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 birch pollen, grass pollen or house dust mite allergic individuals were stimulated with peptide mixes encompassing immunodominant epitopes from corresponding major allergens. After 2 weeks of in vitro amplification, cells were stained with both the appropriate tetramer and antibodies directed to CD25, CD30, CD39, CD69, CD137, CD154, GITR, HLA-DR and ICOS, before FACS analysis. RESULTS: Following allergen stimulation, percentages of tetramer(+) cells among CD4(+) CD154(+) cells range from 5% to 87%, depending upon donors. As for CD154, a large inter-individual variability is observed in terms of surface expression for all activation markers tested in allergen-stimulated PBMCs. T cells reactive with either tetramers (0.4-10.4% CD4(+) T cells) or anti-marker antibodies (2.2-32.7% CD4(+) T cells), but not both, are observed, reflecting the presence of anergic as well as non-specifically activated cells. Tetramer(+) /marker(+) , tetramer(+) /marker(-) and tetramer(-) /marker(+) cells were compared for their capacity to express cytokines, demonstrating that only the former represent bona fide allergen-specific activated CD4(+) T cells, based upon a higher expression of cytokines or corresponding genes in presence of the allergen. CONCLUSION AND CLINICAL RELEVANCE: No strict correlation exists between tetramer staining and the expression of multiple activation markers in stimulated CD4(+) T cells. Dual staining allows to discriminate functional tetramer(+) /marker(+) vs. anergic (tetramer(+) /marker(-) ) allergen-specific T cells or non-specifically activated (tetramer(-) /marker(+) ) T cells. Combining tetramer staining with the detection of activation markers helps understanding patient heterogeneity regarding specific CD4(+) T cell responses. This approach has immediate relevance for monitoring immune changes induced during specific immunotherapy.
BACKGROUND: Major histocompatibility complex (MHC) class II tetramers (tetramers) allow to detect allergen-specific CD4(+) T cells at a single-cell level. Limits to this technology include HLA restriction and the need to identify immunodominant T cell epitopes. OBJECTIVE: Assessing the expression of various activation markers following allergen stimulation to replace tetramer staining. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 birch pollen, grass pollen or house dust mite allergic individuals were stimulated with peptide mixes encompassing immunodominant epitopes from corresponding major allergens. After 2 weeks of in vitro amplification, cells were stained with both the appropriate tetramer and antibodies directed to CD25, CD30, CD39, CD69, CD137, CD154, GITR, HLA-DR and ICOS, before FACS analysis. RESULTS: Following allergen stimulation, percentages of tetramer(+) cells among CD4(+) CD154(+) cells range from 5% to 87%, depending upon donors. As for CD154, a large inter-individual variability is observed in terms of surface expression for all activation markers tested in allergen-stimulated PBMCs. T cells reactive with either tetramers (0.4-10.4% CD4(+) T cells) or anti-marker antibodies (2.2-32.7% CD4(+) T cells), but not both, are observed, reflecting the presence of anergic as well as non-specifically activated cells. Tetramer(+) /marker(+) , tetramer(+) /marker(-) and tetramer(-) /marker(+) cells were compared for their capacity to express cytokines, demonstrating that only the former represent bona fide allergen-specific activated CD4(+) T cells, based upon a higher expression of cytokines or corresponding genes in presence of the allergen. CONCLUSION AND CLINICAL RELEVANCE: No strict correlation exists between tetramer staining and the expression of multiple activation markers in stimulated CD4(+) T cells. Dual staining allows to discriminate functional tetramer(+) /marker(+) vs. anergic (tetramer(+) /marker(-) ) allergen-specific T cells or non-specifically activated (tetramer(-) /marker(+) ) T cells. Combining tetramer staining with the detection of activation markers helps understanding patient heterogeneity regarding specific CD4(+) T cell responses. This approach has immediate relevance for monitoring immune changes induced during specific immunotherapy.
Authors: Carla Oseroff; Lars H Christensen; Luise Westernberg; John Pham; Jerome Lane; Sinu Paul; Jason Greenbaum; Thomas Stranzl; Gitte Lund; Ilka Hoof; Jens Holm; Peter A Würtzen; Kåre H Meno; April Frazier; Veronique Schulten; Peter S Andersen; Bjoern Peters; Alessandro Sette Journal: Clin Exp Allergy Date: 2016-11-07 Impact factor: 5.018
Authors: Giorgio Walter Canonica; Linda Cox; Ruby Pawankar; Carlos E Baena-Cagnani; Michael Blaiss; Sergio Bonini; Jean Bousquet; Moises Calderón; Enrico Compalati; Stephen R Durham; Roy Gerth van Wijk; Désirée Larenas-Linnemann; Harold Nelson; Giovanni Passalacqua; Oliver Pfaar; Nelson Rosário; Dermot Ryan; Lanny Rosenwasser; Peter Schmid-Grendelmeier; Gianenrico Senna; Erkka Valovirta; Hugo Van Bever; Pakit Vichyanond; Ulrich Wahn; Osman Yusuf Journal: World Allergy Organ J Date: 2014-03-28 Impact factor: 4.084
Authors: Josalyn L Cho; Morris F Ling; David C Adams; Lucas Faustino; Sabina A Islam; Roshi Afshar; Jason W Griffith; Robert S Harris; Aylwin Ng; Giorgia Radicioni; Amina A Ford; Andre K Han; Ramnik Xavier; William W Kwok; Richard Boucher; James J Moon; Daniel L Hamilos; Mehmet Kesimer; Melissa J Suter; Benjamin D Medoff; Andrew D Luster Journal: Sci Transl Med Date: 2016-10-05 Impact factor: 19.319
Authors: Karen A Smith; Nicola J Gray; Elizabeth Cheek; Femi Saleh; Jo Lavender; Anthony J Frew; Florian Kern; Michael D Tarzi Journal: BMC Immunol Date: 2013-03-22 Impact factor: 3.615