Isolation of CD4⁺CD25⁺ Regulatory T Cells for Clinical Trials

Efficient enrichment of CD4⁺CD25^highFOXP3⁺ T cells from leukapheresis products by magnetic cell separation under GMP conditions. A, A standard leukapheresis product (No. 6 from Table 1) was depleted of B cells and separated into CD25⁺ and CD25⁻ cell fractions as detailed in the “Materials and Methods” section. At each step of the procedure, aliquots were taken and analyzed by FACS. Plots show the initial leukapheresis product (left panel), the staining pattern of the cells after B-cell depletion and incubation with anti-human CD25 beads (second panel), the CD25-enriched target cell fraction (third panel), and the CD25-depleted negative cell fraction (right panel). B, Cells from the leukapheresis product (left panel), the target cell fraction (middle panel), and the negative cell fraction (right panel) were fixed, permeabilized, and stained for FOXP3. Plots are gated on CD4⁺ T cells within the respective cell populations.

CD4⁺CD25⁺ T cells isolated under GMP conditions show a biphasic expression of CTLA-4 and can be further separated into 2 subpopulations with distinct FOXP3 messenger RNA and protein levels. A, Cell fractions obtained by magnetic cell separation of a leukapheresis product under GMP conditions were analyzed for intracellular CTLA-4 expression. Histograms represent CD4⁺ T cells within the CD25-enriched target cell fraction (CD25⁺) and the CD25-depleted negative cell fraction (CD25⁻) or the CD4⁺CD25^high (CD25^high) and CD4⁺CD25^int (CD25^int) T-cell subpopulations as defined by the sort gates used for further separation of the target cell fraction by FACS. B, Reanalysis of the CD25 expression levels of further FACS-purified CD4⁺CD25^high (CD25^high), CD4⁺CD25^int (CD25^int), and CD4⁺CD25⁻ T cells (CD25⁻). Cells are from the same separation as in (A). C and D, FOXP3 messenger RNA (C) and protein expression levels (D) of the target cell fraction (CD25⁺) in comparison to CD25^high, CD25^int, and CD25⁻CD4⁺ T cells obtained by further FACS purification from the respective cell fractions. Values in (C) represent means + SEM from 4 consecutive isolations.

CD4⁺CD25⁺ T cells isolated by magnetic cell separation under GMP conditions are hypoproliferative and show suppressive activity. CD4⁺ T cells with high, intermediate, or negative expression levels for CD25 were purified by FACS from the target cell fraction and the negative cell fraction. A, Cells from the target cell fraction (CD25⁺) or the various subpopulations (CD25^high, CD25^int, and CD25⁻) were stimulated with anti-CD3 in the presence of autologous antigen-presenting cells for 4 days. Proliferation was determined by ³H-thymidine incorporation and is shown in relation to that of autologous CD4⁺ T_resp cells (see “Materials and Methods” for details). B, Autologous CD4⁺ T_resp cells and cells from the target cell fraction (CD25⁺) or the purified subpopulations (CD25^high, CD25^int, and CD25⁻) were cocultured at the indicated ratios and stimulated as described in (A). Proliferation was determined by ³H-thymidine incorporation, and the percentage suppression was calculated by comparing the proliferative response in the cocultures with cultures of T_resp cells alone, as described in “Materials and Methods.” All assays in (A) and (B) were performed in triplicate. Data represent the means + SEM of all 6 isolations.