An Optimized Method for the Functional Analysis of Human Regulatory T Cells

Abstract

Naturally occurring regulatory T cells (Treg) suppress the activation of antigen‐responsive T cells in a cell contact‐dependent manner. In order to investigate the impact of soluble mediators and receptor–ligand interactions on the interplay between naive T cells and Treg, a reproducible suppressor cell assay which functions in the absence of additional feeder cells or antigen‐presenting cells is mandatory. Here, we describe such a method which is suited to study the modulation of responder T cell/Treg interactions in vitro. Treg were isolated from negatively purified total human CD4+ T cells by positive selection using anti‐CD25 monoclonal antibody (MoAb)‐coated Dynabeads followed by a detachment step. The remaining CD4+ CD25− responder T cells were cocultured with CD4+ CD25+ Treg in the presence of T‐cell Activation/Expansion Beads from Miltenyi Biotec pre‐coated with anti‐CD3 plus anti‐CD28 monoclonal antibody (MoAb). The optimal concentration for coating was 5 μg/ml for both MoAb. At this concentration, strong proliferation of responder T cells was elicited which was almost completely suppressed by Treg at 1:1 cell ratios. When higher concentrations of anti‐CD3/anti‐CD28 MoAb were used for coating, Treg also showed some degree of proliferation. The optimized suppressor assay proved to be highly reproducible and was used here to confirm the partial or complete reversal of Treg‐mediated T‐cell suppression by some cytokines (IL‐2, IL‐15), soluble IL‐6 receptor/IL‐6 fusion protein and recombinant GITR‐ligand. Furthermore, our data confirm that Treg do not need other cell types to suppress proliferation of CD4+ CD25− responder T cells.

Feedback loop of immune regulation by CD4+CD25+ Treg

“…If there is interaction between the two populations that may influence the behavior of Tregs, it would not be appropriate to characterize the behavior of Tregs in this way. However, many investigators followed this way of suppressive assay in which it is impossible to discriminate which population is actually proliferating in the coculture (Larkin et al, 2007;Oberg et al, 2006;Thornton and Shevach, 2000). In this way, as the stimulating signals are increased, Tregs begin to proliferate slightly, so-called, ''loss of anergy,'' along with the loss of suppressive activities, that is, vigorous proliferation in the coculture (Oberg et al, 2006).…”

“…This anergic state is closely linked with suppressive activity as abrogation of anergic state by TCR stimulation with high-dose IL-2 or CD28 ligation results in concomitant loss of suppressive activity (Oberg et al, 2006). Considering their low frequency in the periphery, it may be reasonable to raise the doubt how Tregs in anergic state can regulate the rapidly expanding effector cells properly in a contact-dependent manner when they are triggered.…”

Differential but Direct Abolishment of Human Regulatory T Cell Suppressive Capacity by Various TLR2 Ligands

“…The purity of the selected responder T cells and Tregs was .97%. The isolated T cells were cultured in serum-free X-VIVO-15 medium (Cambrex Bio Science, Verviers, Belgium) in an APC-free suppression assay as described (38). Briefly, 10 4 or 10 5 purified responder T cells, 2.5 3 10 3 or 5-7.5 3 10 4 purified autologous Tregs, or the coculture of these two subsets were stimulated with Activation/Expander Beads (Miltenyi Biotec).…”

“…1A, T) or responder T cells (Fig. 1A, R) were affected by TLR2 ligands, each of the two T cell populations was isolated, separately pretreated for 24 h with the different TLR2 ligands, washed, and analyzed in the suppression assay (38). We observed that the Treg-mediated suppression of responder T cell proliferation (R/T) was completely abrogated after pretreatment of Tregs with a mixture of Pam 2 CSK4, FSL-1, and Pam 3 CSK4 in the majority (four of five) of experiments (representative experiment shown in Fig.…”

Dysfunctional CD4+,CD25+ regulatory T cells in untreated active systemic lupus erythematosus secondary to interferon‐α–producing antigen‐presenting cells

“…CD4ϩ T cells were negatively isolated from fresh PBMCs by magnetic labeling of non-CD4ϩ T cells with a biotin-antibody cocktail and antibiotin MicroBeads (CD4ϩ T Cell Isolation Kit II; Miltenyi Biotec, Sunnyvale, CA) and then passing them over an LD magnetic bead column (Miltenyi Biotec). According to previously described techniques (17), the effluent preenriched CD4ϩ cells were labeled directly with CD25 Dynabeads using 75% of the recommended amount of beads and then separated using a magnetic particle concentrator (both from Dynal Biotech, New Hyde Park, NY). The supernatant contained the CD4ϩ,CD25Ϫ T cells.…”