Cd127 pe

The cells were washed twice with PBS and stained for 30 min at room temperature in the dark with fluorescent-labeled anti-human monoclonal antibodies mixture: CD4-fluorescein isothiocyanate (FITC), CD25-PC5, and CD127-PE (Beckman Coulter, Brea, CA, United States). Quantification beads (Beckman Coulter, Brea, CA, United States) were used to assess absolute counts of cell subsets. Background fluorescence and nonspecific staining were assessed using appropriate isotype- and fluorochrome-matched control monoclonal antibodies. The frequencies and absolute counts of CD4⁺CD25^highCD127^-/low Tregs and the percentage of Tregs on gated CD4⁺ lymphocytes were assessed by a Cytomics FC 500 flow cytometer (Beckman Coulter, Brea, CA, United States).

The ability of expanded Tregs to generate new Tregs infectiously from naïve responder cells was measured using the “Treg-MLR” as described previously^{24 (link)}. Briefly, MLR cultures were set up with “recipient” CFSE-labelled responders stimulated with irradiated (3,000 rads) and PKH26-labelled donor-specific or allo-irrelevant PBMC. PKH26-labelled modulator cells composed of either Tregs or R-PBMC treated in an equivalent manner to serve as controls were then added at modulator: responder ratios of 1:10, 1:50, and 1:250. On day 7, flow cytometry was performed on the cultured cells after labelling with CD127-PE, CD4-ECD, CD25-PC7 (all from Beckman-Coulter), and FOXP3-PC5 (eBioscience). PKH26-labelled modulators and any surviving stimulators as well as CD127-PE⁺ responder cells were gated out, and CD4⁺ cells that proliferated (as determined by reduced CFSE expression) were analyzed for CD25 and FOXP3 expressions. Thus, the percentage of CD4⁺CD127⁻CD25^HighFOXP3⁺ cells that were newly generated in the proliferating responder cells was determined.

The phenotypes of CD8⁺ T-cells in whole blood (using Optylyse, Beckman Coulter, Marseille, France) or isolated cells were distinguished by flow cytometry using multiple antibodies: CD127-PE (5μl, clone R34.34, AB_131301, Beckman Coulter), CD8-FITC/PeCy5 (5μl, clone HIT8a, AB_395996 and AB_395998), CD45RA-APC/PECy5 (3μl, clone HI100, AB_398468 and AB_395881, BD Pharmingen, BD Bioscience, San Jose, CA, USA) and CCR7-APCCy7 (5μl, clone G043H7, AB_10916390, Biolegend, San Diego, CA, USA). Freshly isolated cells (1x10⁵ lymphocytes per sample) were incubated in 1% BSA-PBS (100μl) for 30 minutes on ice, followed by 2 washes with 1% BSA-PBS, protocol adapted from Nascimbeni and Rehermann [37 (link)]. Cell subsets were distinguished as follows: naïve (T_N, CD45RA⁺CCR7⁺), central memory (T_CM, CD45RA^-CCR7⁺), effector memory (T_EM, CD45RA^-CCR7^-), and terminally differentiated effector memory (T_EMRA, CD45RA⁺CCR7^-). When analyzing IH-CD8⁺ T-cells, the flow cytometer was calibrated using blood CD8⁺ T-cells to conserve the number of IH-CD8⁺ T-cells available for study, which revealed a higher degree of autofluorescence in IH-CD8⁺ T-cells compared to blood-derived cells, as previously reported by others [38 (link)]. All flow cytometry analyses excluded dead cells, on the basis of forward and side scatter profiles, and gates were set using the principle of fluorescence minus one (FMO).

For research expansion samples on days 0, 14 and 21 as well as post-transplant immune monitoring, antibodies against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 (all Beckman-Coulter) and FOXP3-PC5 (eBioscience, San Diego, CA) were used. Chemokine and receptor characterizations were performed using CXCR3/GARP-Pacific Blue, CXCR4/CD62L-PerCP-Cy5.5, CCR7/CD45RO-APC-Cy7, CD45RA-Alexa700, and CTLA-4-APC (all from eBioscience, San Diego, CA).
For the GMP expanded Treg products 1–9, three separate panels were used consistently throughout Treg expansion and all antibodies were purchased from Miltenyi. Panel one: CD8-APC, CD20-FITC, CD14/15/56-PE, CD45-Pacific Blue and 7AAD-PerCP-Cy5.5. Panel two: CD25-APC, CD4-FITC, CD127-PE, CD45-Pacific Blue and 7AAD-PerCP-Cy5.5. Panel three: CD4-FITC, CD25-PE, Foxp3-APC and CD45-Pacific Blue. Research grade flow cytometry was performed on a Beckman-Coulter FC500 whereas clinical flow cytometry was done on a BD-LSR, as previously described^{37 (link),54 ,55 (link)}.
Patient post-transplant flow cytometric analyses were done on a Beckman-Coulter FC500 using antibodies (all Beckman-Coulter) against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 and FOXP3-PC5 (eBioscience); IL10-FITC, IgD/M-PE, CD19-ECD, CD27-PC5, CD24-PC7; Helios-FITC, CD3-ECD, CD28-PC5, CD8-PC7 and FOXP3-PE (eBioscience), among others.

As previously described [1 (link), 8 (link), 9 (link)], after Ficoll-Hypaque gradient centrifugation, PBMC were labeled with CFSE following the manufacturer’s instructions (labeling efficiency of >99%). 5x10⁵ CFSE labeled responding PBMC from healthy volunteer (A) were cultured with 5x10⁵ PKH26 labeled stimulator cells from HLA-2DR-matched (Bx) or HLA-mismatched (Ix; indifferent 3^rd party) laboratory volunteers in 48-well culture plates. On days 0 (baseline), 5, 7 and 9 flow cytometric analyses were performed for surface markers using CD4-ECD, CD25-PC7, CD127-PE (all from Beckman-Coulter, Miami, FL) and for intracellular FOXP3-PC5 (eBioscience, San Diego, CA) as previously described [7 (link), 8 (link)]. Gated viable lymphocytes were further gated followed for CFSE bright and dim cells which were negative for both CD127-PE and PKH26 (thus gating out CD127⁺ responders and any residual stimulators). This was followed by gating for CD4⁺ cells. CFSE dilution in these CD4⁺ responders assessed the extent of proliferation, i.e., non-proliferating (CFSE high) or proliferating (CFSE low) cells. The expression of CD25 and FOXP3 were analyzed in the non-proliferating and proliferating populations. Data were calculated as percentage of CD127^-CD4⁺ cells that were CD25⁺FOXP3⁺ (total Tregs) or CD25^HighFOXP3⁺ (natural Tregs; nTregs) [10 (link)].

For “donor” B cell expansion experiments, flow cytometry was performed on cultured PBMC on indicated days using antibodies against CD19-PC7, CD80-PE, CD86-PC5, HLA-DR-ECD (all from Beckman-Coulter, Miami, FL). To phenotype Tregs, antibodies against CD4-FITC, CD127-PE, CD3-ECD, CD25-PC7 (all from Beckman-Coulter) and FOXP3-PC5 (eBioscience, San Diego, CA) were used on days 0, 14 and 21, (also on day 28 in longer-term cultures). All detection was performed on a Beckman-Coulter FC500 flow cytometer as previously described^{9 ,24 (link),25 (link)}. The gating strategy used for the analyses including that for the negative controls is shown in Supplemental Fig. 1.