Depletion of CD45RA T cells: Advantages and disadvantages of different purification methods.

Background: Recently, new advances were made regarding the depletion of CD45RA naïve T cells from haploidentical grafts as they are suspected to be the most alloreactive.

Methods: Within this project we investigated CD45RA-depletion from G-CSF mobilized PBSC by two different purification strategies according to GMP, specifically direct depletion of CD45RA cells (one-step approach), or CD34-positive selection followed by CD45RA-depletion (two-step approach).

Results: With log -3.9 and - 3.8 the depletion quality of CD45RA T cells was equally for both approaches together with a close to complete CD19 B cell depletion. However, due to a high expression of CD45RA the majority of NK cells were lost within both CD45RA depletion strategies. Stem cell recovery after one-step CD45RA-depletion was at median 52.0% (range: 49.7-67.2%), which was comparable to previously published recovery data received from direct CD34 positive selection. Memory T cell recovery including CD4 and CD8 memory T cell subsets was statistically not differing between both purification approaches. The recovery of CD4 and CD8 T cells was as well similar, but overall a higher amount of cytotoxic than T-helper cells were lost as indicated by an increase of the CD4/CD8 ratio.

Conclusions: CD45RA-depletion from G-CSF mobilized PBSC is feasible as one- and two-step approach and results in sufficient reduction of CD45RA T cells as well as B cells, but also to a co-depletion of NK cells. However, by gaining two independent cell products, the two-step approach enables the highest clinical flexibility in regard to individual graft composition with precise dosage of stem cells and T cells.