Initial blood samples were obtained with informed consent under an Institutional Review Board (IRB) approved protocol. Manual muscle testing was performed as previously described.5
Blood samples were obtained from 14 healthy control subjects after obtaining informed consent under an IRB-approved protocol. Control data is being collected for an ongoing study of Treg function in autoimmune MG.
Collection of peripheral blood mononuclear cells (PBMCs)
Blood samples (80cc) were drawn from the patient and control subjects into heparinized tubes, diluted 1:1 with sterile HBSS at room temperature and centrifuged on Ficoll-Paque gradients (GE Healthcare bioscience). PBMC from the interface were recovered, washed in HBSS, counted in a trypan blue dye and used for the experiments.
Antibodies and Cell culture reagents
Allophycocyanin (APC) conjugated anti-human CD4, Phycoerythrin (PE)-conjugated anti-human CD25, Alexa Fluor 488 (AF488) conjugated anti-human Foxp3, phycoerythrin-Cy-7 (PE-Cy7) conjugated anti-human CD127 and their respective Isotype controls were purchased from eBioscience, CA, USA. RPMI 1640 media supplemented with 1% sodium pyruvate, 1% nonessential amino acids, 2mM L-glutamine, 20mM HEPES, 50 U/ml penicillin and 50 µg/ml streptomycin (all from GIBCO, CA, USA), 10% heat inactivated human AB serum (Invitrogen, CA, USA) were used as culture medium. Anti-human CD3 (Clone OKT3) and CFSE were purchased from eBioscience and Invitrogen, respectively. Two different synthetic peptides representing amino acid sequences (aa195–212: DTPYLDITYHFVMQRLPL and aa257–269: LLVIVELIPSTSS) of the human α-subunit of the nicotinic acetylcholine receptor (AChR) were synthesized and HPLC purified (96%) by the Protein Research Laboratory, University of Illinois at Chicago. Peptides were dissolved in DMSO at high concentration (10mg/ml), aliquoted and stored at −80°C.
Sorting of T cell subsets
PBMCs (1×108) were washed once and suspended in 500µl FACS buffer (PBS + 0.1% BSA + 1mM EDTA) in a 5ml polystyrene round bottom tube. After addition of 5µl/107 cell volume APC-CD4, PE-CD25, and PECy7-CD127 antibodies, the cell suspension was mixed gently and incubated at 4°C for 30 min. Then, cold FACS buffer was added and cells were pelleted. Labeled T cells were sorted by using a MoFlo (Becton Dickinson). The sorted gates for the T cell subsets were set to include only those events exhibiting the CD4-specific fluorescence that were also within the lowest density region of a scatter plot. Based on the CD4 gate, cells were further gated based on CD127 and/or CD25 expression [CD4+CD25highCD127low Regulatory T cells (Treg cells) and CD4+CD25− T effector cells (Teff)]. Cells were collected into 100% human AB serum and washed once with media (RPMI 1640 medium with 5% human AB serum) until they were ready to be plated in suppression assay. Sorted Treg and Teff cells were >96% purity. Isotype controls were run to determine the gating parameters.
FOXP3 expression analysis
For CD4+Foxp3+ T cell analysis, 1×106 PBMCS per sample were resuspended in 100µl FACS buffer in a 5ml round bottom polystyrene tube. After addition of 5µl APC-conjugated anti-human CD4, the cell suspension was mixed gently and incubated at 4°C for 30 min. After incubation, staining buffer was added to each sample, and cells were pelleted and resuspended in 100 µl buffer. Then, Foxp3 staining was conducted as specified below.
Intracellular staining was conducted using the recommended procedure obtained from the manufacturer (eBioscience). Briefly, 25,000 sorted Treg cells (CD4+CD25highCD127low) per sample were washed once with FACS buffer and fixed for 60 min using Foxp3 Fixation/Permiabilization buffer (eBioscience, CA, USA). After wash with added FACS buffer, the cell pellet was resuspended with 100µl FACS buffer. 10 µl Alexa fluor 488-conjugated anti-human FOXP3 was added, and cells were lightly vortexed and incubated at room temperature for 30 min. After incubation, FACS buffer was added to each sample, and cells were pelleted, resuspended in 200 µl buffer, and analyzed on a flow cytometer (CyAn ADP, DakoCytomation). Isotype controls were run to determine the gating parameters.
CD4+CD25− T effector cells were resuspended in PBS (0.1% BSA) at 2×106 cells/ml and incubated with CFSE (final concentration: 2 µM) for 10 min at 37 °C. Cells were washed and resuspended in culture medium for 15 min to stabilize the CFSE staining. After a final wash step, cells were resuspended in culture medium at the indicated cell concentrations. Suppression assays were performed as per the method of Venken et al. (2007). Briefly, CFSE labeled CD4+CD25− T cells (responder cells) were cultured in duplicate in 96-well round bottom plates (Falcon, NJ, USA) at 2×104 cells per well in 200µl medium with 1×105 autologous antigen presenting cells (APCs) in the absence or presence equal numbers of CD4+CD25highCD127low/− Tregs (Tresp-Treg ratio 1:0 or 1:1). APCs were prepared from PBMCs by 1-h plastic adherence (to deplete T cells), and the adherent fraction was irradiated with 2000 rad immediately prior to use in the co-culture. Cell cultures were stimulated with 2 µg/ml anti-CD3 (non-specific proliferation) or 3µg/ml AChR peptides (antigen specific proliferation). Cells were also cultured in the presence of non-stimulated irradiated PBMC to assess the background proliferation for each cell type/experiment. After 4 days, cells were harvested, stained for CD4, and the CFSE signal of gated lymphocytes was analyzed by flow cytometry. The suppressive capacity of Tregs towards responder cells in co-culture was expressed as the percentage of proliferation of CFSElow CD4+ T cells. The autofluorescent signal of CFSE unlabeled CD4+CD25hiCD127low T cells (Tresp-Treg ratio 0:1) was subtracted to exclude background levels that would interfere with CFSE diluted cells.