www.dynalbiotech.com12DYNALogue 02/2002Customer ReportCD4+CD25+ cells isolated by Dynabeads display classical regulatory properties in limiting dilution assay of human mixed leucocyte reaction.David S. Game, Maria Hernandez- Fuentes, W. Fai Ng, Afzal N. Chaudhry, Robert I. Lechler.Introduction: There is substantial evidence that immune regulation plays an important part in tolerance to self-antigens and alloantigens in experimental models of autoimmune disease and transplantation 1. The discovery that the CD4+ T cell is essential in this process 2 has focused attention on identifying T cell subsets that are responsible for this effect. Since 1995 there has been accumulating evidence that CD4+ cells expressing the a-subunit of the IL2 receptor (CD25) can act as potent immune response suppressors 3. Most early work was on murine systems but we, and others have shown an analogous population in the peripheral blood of humans 4. Our laboratory has previously refined the use of limiting dilution analysis (LDA) based on the methods of Mehrabi et al. 5 in the study of alloresponses: this technique allows the determination of frequencies of alloreactive cells from peripheral blood 6. Dozmorov et al. have shown theoretically, that with certain assumptions, frequencies for regulatory populations can be calculated from LDA 7. We have further refined these modifications and used LDA to assess whether this technique can be applied to study the regulatory properties of CD4+CD25+ cells.Materials and Methods: Peripheral blood mononuclear cells (PBMC) from healthy volunteers were obtained by density gradient centrifugation over Lymphoprep (Nycomed, Norway). CD4+ T cells were obtained by incubation with different MoAbs: CD8, CD14, CD33 and CD19, CD16 and CD56 (Diaclone, France), followed by negative selection on Dynabeads Goat anti-Mouse IgG magnetic beads (Dynal Biotech, Norway), (4 beads per target cell). This amount was split into 2 aliquots for serial incubations with Dynabeads, each for 30 minutes with a wash in-between. For all washing steps with beads PBS supplemented with 0.5% fetal calf serum (SeraQ, UK) was used. Some CD4+ cells were retained and the remainder subjected to selection by CD25 linked Dynabeads at 2 beads per cell present in 1 ml medium, incubated for 30 minutes (DynalBiotech). Those cells not selected by the Dynabeads onthe magnet (Dynal MPC) were designated CD4+CD25-. The beads were then washed four times and the supernatants discarded. The CD4+CD25+ cells were released from the Dynabeads by mixing with DETACHaBEAD CD4/CD8 (Dynal Biotech) at 10 ml DETACHaBEAD in 100 ml medium per 107 cells at 20C for1 hour. The beads were then removed with a Dynal MPC. Purification efficacy was measured by flow cytometry (fig. 2). All subsequent assays were performed in RPMI 1640 supplemented with Penicillin/Streptomycin, L-Glutamine 2mM (all Invitrogen, UK) and 10% human AB serum (Harlan Sera-Lab, UK). For LDA, replicates of 12 wells at 7 doubling dilutions of responder cells (from 5 x 104 per well) in 100 ml of medium were aliquoted; medium alone was added to the 12 control wells. Irradiated (30 Gy) stimulator PBMCs (5 x 104 in 100 ml) were added to all wells. The different responder populations tested were whole CD4, purified CD4+CD25+, purified CD4+CD25- and co-culture of equal numbers of CD4+CD25+ and CD4+CD25. After 48 hours, 50 ml of supernatant was withdrawn from each well for IL2 estimation . Proliferation assaysAfter 6 days, 50 ml of 1 mCi / ml of Thymidine-H3 was added. Plates were harvested after a further 12 hours. Radioactivity was measured in a scintillation counter (Wallac, Finland). Wells were scored positive when counts were higher than the mean +3 standard deviations of the control wells (irradiated stimulator cells only). IL-2 estimation To each 50 ml of supernatant, 5 x 103 CTLL- 2 in 100 ml of medium were added to each well. The frequency of IL-2 producing cells was obtained by bioassay with the IL-2 dependent CTLL-2 line as described previously 8. Kinetic models For single-hit kinetics the frequencies with confidence intervals were calculated with the modified score function based on a maximum likelihood method. The dual frequency kinetics were based on non-linear least squares method. Both calculations were performed using Excel 7.0 and software written with visual basic (Dr A.N. Chaudhry). For each assay, experimental data is fitted to the function and c2 value of goodness of fit with the corresponding p value are obtained with the program. Frequencies were only recorded if they adjusted to the function with a p 0.05. All frequencies are given as 1 in n number of cells. For the 2 frequency program, several assumptions must be made to derive a curve 7. First, if a given number of regulators are present, they will proliferate but below this number they will not. Second, at a given ratio of regulator to responder there will be suppression but below this ratio