Megakaryocyte Colony-Forming Units (CFU-Meg) Growth Is Enhanced by Alemtuzumab Mediated Lymphocyte Depletion: In Vitro Experiments and a Case Report of Acquired Amegakaryocytic Thrombocytopenic Purpura.
Authors: Doubek Michael , Koristek Zdenek , Smardova Lenka , Mayer Jiri 
Published: April 2020

Abstract
Background. Alemtuzumab (Ale) induces destruction of CD52 antigen-bearing cells by antibody-dependent cell-mediated lysis, complement-mediated lysis, and induction of apoptosis. As a potent T cell depleting agent, Ale is used also for GvHD prophylaxis, given either i.v. around the time of transplantation, or directly "in the bag" to the cell suspension containing hematopoietic stem cells and lymphocytes. Contrary to bone marrow-derived grafts, the experience with addition of Ale to the peripheral blood progenitor cells (PBPC) suspension is limited. Therefore, we decided to study the effect of short-time incubation of PBSC samples with Ale using flow-cytometry and cultivation assays.
Methods. After an informed consent, a sample of PBSC product (15 mL) was obtained from the collection bag immediately after a leukapheresis (COBE Spectra) in 8 healthy donors mobilized by G-CSF. Cells were washed using 4% human albumin and then incubated at 37°C with 1) Ale at the dose of 5 μg/106 cells, and 2) water for injection as a control. Autologous plasma as a source of complement was added after 15 min of incubation and cells suspensions were further incubated for 15 minutes. Both cell suspensions were then washed, resuspended in 4% human albumin, and samples were collected for blood count, flow-cytometric analysis, and cultivation assays (CFU-GM, BFU-E, CFU-Meg).
Results. As expected, Ale caused profound T-cell (by a median of 99.3% vs. 12.3% in control) as well as marked B-cell (by a median of 90.6% vs. 8.3% in control) depletion. CD4+ and CD8+ lymphocytes were eliminated equally by a median of 99.5%, and 99.1%, respectively. Ale also produced a moderate reduction of CFU-GM and BFU-E (by a median of 37.7% vs. 3.3%, and 49.7% vs. 10.8%, respectively). NK-cells were not significantly affected as well as granulocytes and monocytes. Surprisingly, incubation with Ale clearly enhanced the growth of CFU-Meg; in median 8-times more CFU-Meg were observed in samples after incubation with Ale comparing to control samples (from 3 to 21-times, p&lt;0.01). Based on these findings we decided to use Ale for the treatment of 63-year-old woman suffering from severe and therapy resistant (steroids, cyclosporine A) acquired amegakaryocytic thrombocytopenic purpura (AATP). Patient’s bone marrow samples were first tested as described above and a remarkable enhancement of CFU-Meg growth (106 CFU-Meg/1 ml of bone marrow) was observed in a sample incubated with Ale comparing to a control sample not incubated with Ale (2.4 CFU-Meg/1 ml of bone marrow). The patient subsequently received Ale at the dose of 10 mg 3 times weekly for 3 weeks. Ale administration had to be temporarily discontinued for CMV reactivation; however, platelets count significantly increased from 10x109/L to 50x109/L.
Conclusion. Our results demonstrate that megakaryopoiesis is physiologically suppressed by lymphocytes. To the best of our knowledge, this is the first description that profound lymphocyte depletion by Ale enhances megakaryopoiesis in vitro and that Ale can also be used in vivo for the treatment of AATP.