Novel Serial Positive Enrichment Technology Enables Clinical Multiparameter Cell Sorting

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve – especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4high/CD25high/CD45RAhigh ‘regulatory T cells’ and CD8high/CD62Lhigh/CD45RAneg ‘central memory T cells’, have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research

Serial magnetic cell enrichment of naturally occurring regulatory T cells.

<p>(a) Serial positive magnetic enrichment of CD4⁺CD25⁺CD45RA⁺ regulatory T cells (nTregs) from PBMCs. For pre-selection of CD4⁺ cells, PBMCs were first incubated with anti-CD4 Fab-multimers conjugated with <i>Strep</i>-Tactin-functionalized magnetic beads. The resulting positive fraction was then liberated from surface-bound label by D-biotin treatment and washed to remove anti-CD4 reagents. The second purification step comprised the selection for CD25 positive cells from the pre-selected CD4⁺ cell pool via specific anti-CD25 Fab bound to <i>Strep</i>-Tactin coated magnetic beads. Cell bound reagents were again removed from the resulting positive fraction by addition of D-biotin. In a third purification step, CD45RA⁺ cells were isolated from the enriched CD4⁺CD25⁺ cell population by using CD45RA-specific Fab-multimers conjugated to <i>Strep</i>-Tactin-coated magnetic beads. Living lymphocytes in the respective fractions of each selection step are shown. One representative experiment from five independent blood donors is shown. (b) Intracellular FoxP3 staining of triple positive enriched CD4⁺CD25⁺CD45RA⁺ regulatory T cells. (c) Overlay of the enriched CD4⁺CD25⁺CD45RA⁺ cell population (black dots) derived from serial magnetic selection as shown in (a) and the corresponding starting population (underlying grey dots). (d) Summary of cell purities obtained within each purification step of multiparameter magnetic bead-based nTregs purifications as performed in (a) with PBMCs derived from 5 different blood donors (left graph, mean values are indicated). In the right graph, yields (in %) of the target nTregs are shown; mean value is indicated. For all samples analyzed by flow cytometry, at least 50.000 events have been acquired.</p

Serial magnetic cell enrichment of central memory T cells.

<p>(a) Serial magnetic enrichment of CD8⁺CD62L⁺CD45RA^neg central memory T cells from fresh PBMCs. Cells were first incubated with anti-CD8 Fab-multimers conjugated with <i>Strep</i>-Tactin-functionalized magnetic beads in order to pre-select CD8⁺ cells. The resulting positive fraction was then treated with D-biotin and washed to remove all anti-CD8 reagents. In a second step, CD62L positive T cells were enriched from the pre-selected CD8⁺ T cell pool via specific anti-CD62L Fab bound to <i>Strep</i>-Tactin coated magnetic beads and subsequently liberated from the selection reagents as described above. In a final step CD45RA⁺ cells were depleted from the pre-enriched CD8⁺CD62L⁺ cell population using CD45RA specific Fab-multimers conjugated to <i>Strep</i>-Tactin-coated beads. Living lymphocytes in the respective fractions of each selection step are shown. One representative experiment from five independent blood donors is shown. (b) Overlay of the enriched CD8⁺CD62L⁺CD45RA^neg cell population (black dots) derived from serial magnetic selection as shown in (a) and the corresponding starting population (underlying grey dots). (c) Summary of cell purities obtained within each purification step of multiparameter magnetic bead-based T_CM purifications as performed in (a) with PBMCs derived from 5 different blood donors (left graph, mean values are indicated). In the right graph, yields (in %) of the target T_CMs are shown; mean value is indicated. For all samples analyzed by flow cytometry, at least 50.000 events have been acquired.</p