Dr. Patricia Quade-Lyssy (Frankfurt a. M./ DE), Dr. Eliza Wiercinska (Frankfurt a. M./ DE), Prof. Dr. Dr. Halvard Bönig (Frankfurt a. M./ DE; Seattle, WA/ US), Prof. Erhard Seifried (Frankfurt a. M./ DE), Dr. Julia Dzionek (Bergisch-Gladbach/ DE), Dr. Valeriya Olevska (Bergisch-Gladbach/ DE)
Background
In vitro or in vivo depletion of alloreactive T-cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). We established and validated a TCRαß/CD19 depletion module for automatic cell-processing on CliniMACS Prodigy, and investigated quality attributes of the HSPC products. The process was combined with aCD45RA depletion module as such a product could serve as allo-reactivity attenuated donor lymphocyte infusion (DLI) after transplantation.
Methods
We evaluated 6 apheresis products from G-CSF-mobilized volunteer donors which were split, one portion each depleted of CD45RA+ or of TCRα/ß+ and CD19+ cells. Products were assessed for recovery of HSPCs and certain mature subsets, as well as depletion of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 4 °C as well as freeze-thawing on product viability and recovery of WBC and CD34+ cells were assessed by flow cytomerty.
Results
Ten sequential depletions were technically uneventful, proceeding automatically with minimal hands-on time. Depletion of CD45RA+ resp. TCRα/ß+ and CD19+ cells was nearly complete, at least equally to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγ/δ+ and NK cells and even were passively enriched. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions proceeded efficiently. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90 %. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70 % and in line with expectation.
Conclusion
A closed, GMP-compatible process on CliniMACS Prodigy generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαβ/CD19-depleted products included stem cells, TCRγ/δ+ and NK cells. CD45RA-depleted DLI and/or TCRαß/CD19-depleted HSPC products are predicted to be effectively depleted of GvH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.
Offenlegung Interessenkonflikt:
VO and JD are employees of Miltenyi Biotec, manufacturers of the technology described here. HB has received research funding and has served on the speaker"s bureau of Miltenyi Biotec. Studies were supported by in-kind contributions of Miltenyi Biotec. None of the other authors have conflicts to declare.
Funding: Miltenyi Biotec contributed all Miltenyi Biotec reagents and consumables used for these studies.