Ex Vivo Monitoring of Antigen-Specific CD4⁺T Cells after Recall Immunization with Tetanus Toxoid

Abstract

To monitor antigen-specific CD4؉ T cells during a recall immune response to tetanus toxoid (TT), a sequential analysis including ex vivo phenotyping and cytokine flow cytometry, followed by cloning and T-cell-receptor (TCR) spectratyping of cytokine-positive CD4 ؉ T cells, was performed. Grossly, twice as many TT-specific CD4 ؉ T-cell clones, ex vivo derived from the CCR7 ؉ subsets appears to target the bulk of antigen-specific T cells and to reach an analytical power sufficient to adequately delineate in field trials the profile of the antigen-specific response to vaccine.Phenotypic characterization of antigen-specific CD4 ϩ T cells with distinct functional properties represents currently a major challenge in human applied experimental immunology. Ex vivo analysis of these cell populations is critical, since in vitro culture may alter composition and functional properties of the populations of interest. If major histocompatibility complex (MHC) class I tetramer/peptide complexes did greatly facilitate the analysis of antigen specific CD8 ϩ T cells, the use of MHC class II tetramer/peptide has been hampered by the complexity of MHC class II tetramer production, by the usually low CD4 ϩ T-cell precursor frequency, by an often-degenerated peptide epitope specificity, and by non-MHC class II moleculerestricted allelic expression (12,15,24,35,36). As an alternative approach to circumvent these difficulties, we here applied cytokine flow cytometry (9, 11), now extensively used to define ex vivo frequencies and phenotypic and functional characteristics of viral antigen-specific CD4 ϩ T cells (8,22,43), coupled to T-cell-receptor (TCR) spectratype analysis. Indeed, characterization of the unique complementary determining region 3 (CDR3) sequence expression of CD4 ϩ (or CD8 ϩ ) T-cell clones appears to be a sensitive method for monitoring qualitative and quantitative T-cell responses during natural infection or vaccination (21,23,45,48).In the present study, tetanus toxoid (TT) was used as a model vaccine antigen that likely shares common functional characteristics with other nonviral antigens, such as a low frequency of specific CD4 ϩ T cells in the periphery at steady state and a specific CD4 ϩ T-cell response to multiple epitope peptides presented by MHC class II molecules (16,36,40). Using ex vivo cell phenotyping and cloning in combination with TCR spectratype analysis of cytokine-positive CD4 ϩ T cells, we sought to establish experimental conditions to track ex vivo antigenspecific CD4 ϩ T cells in conditions that are likely to reflect clinical situations such as vaccination, nonviral pathogen infections, or allergic diseases.

IL-2 Producing Memory CD4+ T Lymphocytes Are Closely Associated with the Generation of IgG-Secreting Plasma Cells

“…This is distinct from HBsAg and tetanus toxoid, both vaccination Ags and both cleared upon re-encounter. In agreement with our findings, a preferential expansion of the Tcm compartment as compared with the Tem subset was observed upon reencounter of the Tetanus Toxoid (33).…”

Hepatitis B specific T cell immunity induced by primary vaccination persists independently of the protective serum antibody level

“…5). What is not understood is why we did not find a greater expansion of EM and TD cells after antigenic stimulation as other authors did, albeit in different experimental settings [28][29][30][31]. In our experimental setting, the analysis of memory subsets was performed in all T cells and not only in activated/proliferating ones.…”

A guide to measuring phagosomal dynamics

“…Widely used procedures involve protein‐coated polystyrene beads as phagocytic cargo and antibodies that recognize peptide:MHC‐II complexes such as the pair Eα 52‐68 :I‐A b / YAe antibody and others more recently developed [122]. Indirect readouts for MHC‐II presentation include monitoring the cell surface expression of activation markers on T‐cell clones specific to certain peptide:MHC‐II complexes, such as murine OT‐II (reactive to OVA 323‐339 :I‐A b ) [123] or 1H3.1 (reactive to Eα 52‐68 :I‐A b ) [124] T cells, or tetanus toxoid‐specific human T cells [125]. The use of OVA or viral protein‐expressing bacteria such as Escherichia coli , Listeria monocytogenes, or Salmonella typhimurium (STm) [126–128] as phagocytic cargo, followed by the coculture with antigen‐specific T‐cell clones, is also possible.…”

“…Activation of TFEB and TFE3 can also be monitored by assessing their phosphorylation status and nuclear translocation by cellular fractionation and immunoblotting [113]. [123] or 1H3.1 (reactive to Ea 52-68 :I-A b ) [124] T cells, or tetanus toxoid-specific human T cells [125]. The use of OVA or viral proteinexpressing bacteria such as Escherichia coli, Listeria monocytogenes, or Salmonella typhimurium (STm) [126][127][128] as phagocytic cargo, followed by the coculture with antigen-specific T-cell clones, is also possible.…”