Alemtuzumab but Not ATG Decreases Natural Killer (NK) Cell Activity towards Tumor Targets in the Early Phase after Allogeneic Stem Cell Transplantation.

Background: In vivo T cell depletion with ATG or Alemtuzumab is effective to reduce the incidence of graft-versus host disease (GVHD) caused by alloreactive T cells. However, there is also a potential impact of these substances on the function of natural killer (NK) cells who are the predominant cells in peripheral blood in the early phase after hematopoietic stem cell transplantation (HSCT) and mediate beneficial graft-versus-tumor activity. Using a novel flow cytometric assay, which detects the lytic granule membrane protein CD107a as a marker for NK cell degranulation, we investigated the effect of T cell depletion with ATG and Alemtuzumab on NK cell function in the early phase after HSCT.

Methods: PBMCs of 34 patients (pts) at day +30 after allogeneic HSCT and of 16 healthy donors were coincubated at 37°C for 3 h with the NK sensitive cell line HL60. In each tube, containing 400μl effector/target suspension (2x10⁶ cells), 20μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody was added prior to incubation. After the first 1 h 10μl of the secretion inhibitor 2 mM monensin was added. At the end of coincubation cells were stained with mAbs (CD56, CD3) for flow cytometry. The percentage of CD107a expressing NK cells was assessed and the absolute number of degranulating NK cells/μl was calculated.

Results: Treatment Characteristics: Fourteen pts received ATG, ten pts were treated with Alemtuzumab and ten patients did not receive T cell depletion. The source of donor was: MRD 12 and MUD 22. NK cell count: The median NK cell count was: 250/μl in healthy individuals, 250/μl in pts without T cell depletion, 400/μl in pts with ATG and 100/μl in pts receiving Alemtuzumab (p<0.0005; Kruskal-Wallis test). NK cell activity: The median percentage of degranulating NK cells was 5.4% in healthy donors, 4,4% without T cell depletion, 2,8% when ATG was used and 0,8% when Alemtuzumab was given (p<0.0005). The absolute number of CD107a+ NK cells in response to the standardized tumor targets accounted for 13,4/μl (median) in normal donors, 12,9/μl in pts without T cell depletion, 7,6/μl in pts with ATG and 0,9/μl in pts with Alemtuzumab (p=0.001). The percentage and absolute number of CD107a+ NK cells were not significantly different between patients receiving ATG and patients not receiving T cell depletion (p=NS).

Conclusion: With a new and feasible method we were able to quantify and characterize tumor reactive NK cells after HSCT. We found that NK cell mediated cytotoxicity towards tumor targets is influenced by the type of T cell depletion: The NK cell activity in patients receiving Alemtuzumab was considerably reduced whereas ATG had only moderate impact on the NK cell activity in the early phase after HSCT.