CD4+CD25+CD127dim/– Treg isolation with MACS technique
Three cell surface markers include CD4, CD25 and CD127 or two cell surface markers include CD4, CD25 along with one intracellular marker (FOXP3) can be used to isolate Tregs from PBMC. In the current protocol, due to the more feasibility, cheaper, and ease of work, using cell surface markers for Treg isolation is recommended14.
The CD4+CD25+CD127dim/–Treg isolation is performed in a two-simple step. First, labeling the non-CD4+ andCD127high cells using magnetically labeled antibodies and second, using anti-biotin monoclonal antibodies conjugated to Micro Beads. The steps are as follows:
1. Isolation of PBMCs using Ficoll. Dilute the blood sample at 1:1 volume ratio with the appropriate medium (or PBS). Add a volume of density gradient medium to a fresh tube according to the specifications for that density gradient medium. Then, gently layer the diluted blood on top of the density gradient medium. Centrifuge for 30 min at 400g with the break off. Aspirate plasma and platelets on the upper layer using a sterile pipette to avoid platelet contamination. harvest the cells by inserting he pipette directly through the upper plasma layer to the mononuclear cells at the interface. Finally, wash the harvested cells twice in the appropriate buffer for 5 min at 300g. The cells are now ready for cell separations15, 16.
2. Treg cell separationusing MACS column, step1.In the first place, the cell number should be determined and the cell suspension should be spun at 300 g for 10 min followed by aspiration of the supernatant. Then, the cell pellet should be resuspended in 40 µL of MACS buffer (PBS with 2mM EDTA and 0.5% BSA) per 10⁷ total cells. 10 µL of CD4+CD25+CD127–biotinylated antibody per 10⁷ total cells should be added followed by incubation for 5 min in 2−8 °C. In the following, 20 µL of Anti-Biotin microbeads and 30 µL of MACS buffer per10⁷ total cells should be added followed by second incubation for 10 min in 2−8 °C. Before Applying the cell suspension onto the LD MACS column, the column should be rinsed using 2 mL of MACS buffer. In this step, pass-through cells should be collected after adding the mentioned cell suspension onto the column.The cells resulting from this step are unlabelled CD4+ cell fractions17, 18.
3. Treg cell separation using MACS column, step2. Centrifuge the cells obtained from the previous step at 300 g for 10 min and remove the supernatant completely. In the following, the cell pellet should be resuspended in 90 µL of MACS buffer per 10⁷ total cells. At this step, 10 µL of CD25 Beads per 10⁷ total cells should be added followed by incubation for 5 min in 2−8 °C. at the last step, cells should be washed once by adding 1 mL of MACS buffer and centrifuge for 10 minutes at 300 g and supernatant should be discarded immediately. before adding the washed cells to the MS column, cell pelleted should be resuspended in 0.5 mL of MACS buffer per 108 total cells. Before Applying the cell suspension onto the MS MACS column, the column should be rinsed using 0.5 mL of MACS buffer. in this step, cell suspension should be added to the pre-washed MS column. flow-through obtained from this step is containing unlabelled cells19, 20.
Remove the MS column from the stand and place it on sterile 15 mL conical falcon tubes. now to separate magnetically labelled cells from the MS column, 1 mL of MACS buffer should be added onto the MS column and the plunger must be pushed Immediately and firmly. The flow-through obtained at this stage contains the Tregs we are looking for.
4. Flowcytometry analysis of isolated CD4+CD25+CD127dim/– Tregs
Isolated cells should be washed with fresh and complete TEXMACS medium at 1500 rpm for 5 min at 4°C and the supernatant should be discarded. Single cell suspensions should be incubated with 2 µL of FITC-conjugated anti-CD4, 2 µL of APC-conjugated anti-CD25 and 2 µL of PE -conjugated anti-CD25 per 106total cells for 30 min on the ice (or in the 4°C).
After incubation period, stained cells should be washed twice with 500 µLof flowcytometry buffer (PBS with 1% BSA and 0.1% sodium azide) at 1500 rpm for 5 min at 4°C and the supernatant should be discarded. At the end, 500 µLof flowcytometry buffer should be added and data should be collected with flow cytometry(Figure 3A and 3B). Delays of more than 24h may increase the chance of obtaining false-negative or false-positive hence, we recommend fixing the cells using 4% paraformaldehyde solution (diluted in PBS) and storing the fixed cells at 2 to 8°C.
5. Fast and simple protocolfor TregExpansionInvitro
Tregs isolated from the PBMC of beta-thalassemia major kids expanded rapidly with comparable fold-expansion during the 5 days of culture. At day 0, isolated Tregs should be cocultured with 50-100 IU/ml pen-strep, 1mM Sodium pyruvate (C3H3NaO3), 25 mMHepes, 100nM rapamycin, 500 IU IL-2, and 10% human AB serum (or FBS) in TEXMACS medium21. Isolated Tregs should be also activated with anti-CD3/CD28 coated beads at a 4:1 bead:cell ratio at day 0 .Fresh culture medium containing 500 IU/ml IL-2 should be added at day 3 and day 5 post-expansion.Flow cytometric analysis should be performed on harvested cells at day 5 post-expansion. Briefly, cells should be washed and stained with the mentioned antibodies in part 4 for 30 min at 4°C. Appropriate isotype control antibodies should be used for each sample. Following staining, cells were examined by flow cytometry (Figure 3C, 3D and 3E).
6. Functional assay of expanded Tregs
We took advantage of Treg functional (suppression) assays to evaluate the functional effect of expanded Tregs on responding T cells (Tresp). This assay was performed by co-culturing the responding population with the in-vitro expanded Tregs.Responder T cells were obtained from PBMCs using MACS by negative selection.
For assessing the proliferation of Tresp in the presence of expanded Tregs, Tresp should be labeled using carboxyfluoresceinsuccinimidyl ester (CFSE) dye in the first place. For this purpose, isolated Tresp should be washed and the pellet should be resuspended in 1ml PBS containing 0.1 % BSA per 2x106 total cells. 1µL of 5 mM CFSE dye should be added followed by incubation in a dark place at room temperature (RT) for 15 min. After the incubation period, 10 ml of cold complete culture medium should be added and the tube should be incubated again on the ice for 5 min. In the next step, cells should be washed with a complete culture medium twice to discard unattached and additional CFSE. Now, labeled Tresps are ready for suppression assay22.1x105 per well of Tresp should be co-cultured with Tregs at different ratios (Treg: Teff = 2:1 and 8:1) in TEXMACS medium supplemented with 10% FBS and in the presence of anti-CD3/CD28-coated beads at a 4:1 bead:cell ratio in 24-well flat-bottom plates.Cells should be incubated at 37°C in the presence of 5% CO2 and 98% O2 for 5 days. After harvesting the cells, the proliferation of CFSE-labelled Tresps should be determined using flow cytometry (Figure 3J).
7. Treg plasticity in the inflammatory environment is the most challenging aspect
Treg plasticity and their conversion to producing inflammatory cytokine cells in the presence of pro-inflammatory cytokines are the major concerns in Treg immunotherapy.Plastic Tregs have several features of Th cells including the expression of specific transcription factors in Th cells and the secretion of Th-related cytokines, while still maintaining the expression of specific transcription factors for Treg23, 24.Hence, the plastic differentiation of Tregs not only shows an association with changes in the stability of Tregs but also increases the complexity of the immune circumstances under inflammatory conditions, especially autoimmune diseases. Signalling through IL-1 receptor and IL-6 receptor in combination with signalling through TGF-β receptor and TCR engagement, induces strong expression of STAT3 (Signal Transducer and Activator of Transcription 3) and RORγT (Retinoic acid-related orphan receptor gamma T) transcription factors which drive the differentiation of conventional T cells to a Th17 lineage in inflammatory conditions22. The capacity of Tregs to adopt a Th1 phenotype has been also demonstrated in murine models25. Nevertheless, the plasticity of the human Treg compartment is still uncharacterized. Moreover, it is not clear to what extent cytokine production by Tregs is due to true plasticity rather than to heterogeneity in the population. Hence, assuming that naive Tregs are capable of upregulating the expression of Th17-associated IL-17 in response to inflammatory cytokines, we simulated inflammatory conditions for expanded Tregs in the culture medium. For this purpose, already expanded Tregs were cultured for 72 h in a medium containing 10 ng/ml IL-1β, 4 ng/ml IL-6, 5 ng/ml TGF-β and 10 IU/ml IL-2 with or without 500 nM rapamycin (Figure 3F-3I). After harvesting the cells, intracellular staining for IL-7 was done as follows:
After harvesting the Tregs, a single-cell suspension at a concentration of 1 x 106 cells/mL in cell straining buffer(PBS containing 2% FBS) should be prepared. For the fixing step, 1 mL fixation buffer (PBS containing 1% paraformaldehyde) per 1 x 106 cells should be added followed by incubation for 15 min at RT. After Centrifuging at 500 g for 5 min at RT, supernatant should be discarded to remove the fixation buffer and a second and third wash of fixed cells with cell staining buffer should be done at 500 g for 5 min.
For permeabilizing the cells, fixed cells should be resuspended in 1 mL permeabilization buffer (0.2 % Tween) per 1 x 106 cells and centrifuge at 500 g for 5 minutes at RT followed by removing the supernatant. In the following، fixed and permeabilized cells should be washed twice with 0.1 % Tween.
For the staining step, in the first place, the primary antibody should be Diluted with permeabilization buffer for an optimal working concentration. Fixed and permeabilized cells should be resuspended in PBS containing 2% FBS, 0.1% Tween, and 2µL of PE-conjugated anti-IL-17 antibody solution followed by Incubate for 20 min on the ice (or at 4°C). After the incubation period, the cells must be centrifuged at 500 g for 5 min at 4°C followed by discarding the supernatant and washed twice with 0.1 % Tween (or permeabilization buffer). As the last step, fixed, permeabilized, and intracellular stained Tregs should be resuspended in 500 uL of cell staining buffer for final analysis.The intracellular expression of IL-17 should be determined using flow cytometry.