Analysis of Factors Influencing Natural Killer (NK) Cell Activity during the Early Phase after Allogeneic Stem Cell Transplantation (SCT).

Background: After allogeneic SCT, NK cell mediated cytotoxicity is an important defense mechanism against residual tumor cells and viral infections. 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 in vivo T cell depletion and the type of conditioning on NK cell function in the early phase after transplantation.

Methods: At day +30 and day +90 after allogeneic SCT with regular (n=14) and dose reduced conditioning (n=8), PBMCs were coincubated at 37°C for 3 h with the NK sensitive cell line HL60. 20μl of PE-Cy5 conjugated anti-CD107a monoclonal antibody (moAb) was added to each tube containing 400μl effector/target cell suspension (2x10⁶ cells, E:T ratio 1:1) prior to incubation. After 1 hour, 10μl of monensin (2mM) was added. After incubation for 3 hours, the cells were stained with conjugated moAb (CD56, CD16, 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 were compared to values from 15 healthy controls.

Results: Twenty two patients (pts.) were investigated. Fourteen pts. received a conventional conditioning regimen and eight a reduced intensity conditioning. T cell depletion was applied in 15/22 pts. (ATG n=12, alemtuzumab n=2, 1 OKT-3 n=1). The type of donor included MRD (n=7) and MUD (n=15). At day +30, the proportion of NK cells with cytotoxic activity (indicated by the mean percentage of degranulating CD107a⁺/CD56⁺ cells) was significantly reduced as compared to normal donors (2.6% vs. 5.6%, p<0.001). At day +90 the percentage of degranulating NK cells was still decreased compared to normal (3.5%, p=0.007). The predominant proportion of degranulating cells was in the CD56^dim/CD16⁻ subpopulation (mean 9.8%). After conventional conditioning, the mean percentage of CD107a⁺ cells was 1,9% at day +30, compared to 4,0% in patients with reduced intensity conditioning (p=0.21). The absolute number of degranulating NK cells was significantly reduced after conventional conditioning (4.1/μl vs. 19.8/μl, p=0.011). Interestingly, we found no influence of in vivo T cell depletion with ATG on the mean value for CD107a⁺ cells at day +30 (2.5% vs. 2.9%, p=0.77).

Conclusion: Although the proportion of NK cells is increased after allogeneic SCT, our data suggest that the cytotoxic activity of these cells is considerably reduced. The absolute number of NK cells with cytotoxic activity is significantly higher after reduced intensity conditioning which may contribute to the effectiveness of these regimens. Antibody induced in vivo T cell depletion with ATG showed no impact on NK cell activity during the first two months post SCT.