Foxp3 pc5

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.

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.

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)].