Antigen-Reactive T Cell Enrichment for Direct, High-Resolution Analysis of the Human Naive and Memory Th Cell Repertoire

Abstract

Ag-specific CD4+ T cells orchestrating adaptive immune responses are crucial for the development of protective immunity, but also mediate immunopathologies. To date, technical limitations often prevented their direct analysis. In this study, we report a sensitive flow cytometric assay based on magnetic pre-enrichment of CD154+ T cells to visualize rare Ag-reactive naive and memory Th cells directly from human peripheral blood. The detection limit of ∼1 cell within 105–106 permitted the direct enumeration and characterization of auto-, tumor-, or neo-Ag–reactive T cells within the naive and even memory CD4+ T cell repertoire of healthy donors. Furthermore, the analysis of high target cell numbers after pre-enrichment of rare Ag-specific T cells from large blood samples dramatically improved the identification of small subpopulations. As exemplified in this work, the dissection of the Ag-specific memory responses into small cytokine-producing subsets revealed great heterogeneity between pathogens, but also pathogen-related microsignatures refining Th cell subset classification. The possibility to directly analyze CD4+ T cells reactive against basically any Ag of interest at high resolution within the naive and memory repertoire will open up new avenues to investigate CD4+ T cell–mediated immune reactions and their use for clinical diagnostics.

Identification of Immunogenic Antigens from Aspergillus fumigatus by Direct Multiparameter Characterization of Specific Conventional and Regulatory CD4+ T Cells

“…Further analysis of cytokine production and phenotype revealed no major differences in the T cells stimulated with the different A. fumigatus morphotype lysates; as shown in Fig. 1D, with all lysates a high frequency of TNF-a and IL-2 producers could be detected, whereas production of the lineage-defining cytokines IFN-g and IL-17 was typically ,10%, although IFN-g production was clearly predominant over IL-17, as described previously (12,30,31). Similarly, irrespective of the A. fumigatus lysate used for stimulation, a comparable number of reactive CD45RO + memory T cells was detected (Fig.…”

“…+ T cells specifically reacting against A. fumigatus can be identified using CD154 expression as a specific read-out for Agactivated CD4 + T cells after short in vitro stimulation with fungal lysate (12,22). To analyze against which A. fumigatus morphotype the human T cell response is directed, PBMCs from healthy donors were stimulated for 7 h with crude lysates from RC, SC, GC, or mycelia containing the total soluble fraction of the mechanically disrupted fungal cells.…”

“…For analysis of cytokine expression, 1 mg/ml brefeldin A (Sigma-Aldrich) was added for the last 2 h of stimulation. Surface staining was performed on the first column, followed by fixation, permeabilization (Inside Stain Kit; Miltenyi Biotec), and intracellular cytokine staining on the second column, as described (12), or staining of Foxp3 using the Foxp3 Staining Buffer Set (Miltenyi Biotec).…”

“…We recently described a highly specific and sensitive assay to enumerate and characterize Ag-specific CD4 + T cells directly ex vivo based on CD154 + pre-enrichment (Ag-reactive T cell enrichment [ARTE]) (12,22). We used ARTE to systematically quantify and characterize the very rare human peripheral CD4 + T cells reacting against crude lysates of A. fumigatus, as well as selected proteins or protein fractions.…”

Peptide immunotherapy in experimental autoimmune encephalomyelitis

“…Can we identify these cells? Flow cytometric approaches using peptide-MHC tetramers, [86,87] or cell surface changes in response to brief in vitro stimulation with antigen [88] suggest that this will become increasingly possible. Can we purify sufficient numbers of autoantigen-responsive cells to allow transcriptional/epigenetic analyses?…”