NKG2C+NKG2A-CD56dim Subset of NK Cells Show Increased Anti-Leukemia Potential in Presence of CTLA4Ig in-Vitro and Is the Key Determinant of Early Relapse and Long-Term Disease-Free Survival without Gvhd Following CTLA4Ig-DLI Based Haploidentical HCT

Abstract

<h3>Background & Methods</h3> CTLA4Ig-primed donor lymphocyte infusions (CTLA4Ig-DLI) following haploidentical HCT were earlier shown to be associated with early proliferation of CD56dim NK cells with lower incidence of disease relapse. We prospectively studied the reconstitution of NKG2C+ subset of NK cells in 60 patients with advanced leukemia (myeloid-28/lymphoid-32), along with functional assays. Conditioning was Flu-Bu-Mel followed by CTLA4Ig-DLI on days +7, +21 and +35. GVHD prophylaxis was PTCy followed by cyclosporine for 60 days [BBMT,2019]. All donors and recipients were CMV seropositive. <h3>Results</h3> All patients engrafted with incidences of grade 2-4 acute and chronic GVHD being 12% and 19.8% respectively. NRM was 5% with an overall survival (OS) of 86.5% at a median of 26 months. Disease progression (DP) was 17.6% between days+28 to +144 (median 90 days). This tended to be lower in patients with myeloid leukemia (7.7% vs 26.3% in lymphoid; p=0.06) and NK-KIR B haplotype (10.9% vs 36.6%, p=0.03). Higher CD56dim NK cells at days+30 (135 vs 17 cells/µl), +60 (150 vs 19 cells/µl) and +90 (121 vs 33 cells/µl) (p< 0.01) without any difference in T cell subsets correlated with lack of DP. NKG2C+/NKG2A- subset of CD56dim NK cells were significantly higher at days +30 (5.3% vs 1.0%), +60 (18.5% vs 1.4%) and + 90 (30.6% vs 2.1%) in those without DP (p <0.0001). The surge of NKG2C+ NK cell subset was independent of CMV reactivation and was sustained at a mean value of 27.2%, 32.8%, 34.5% and 28.1% at 6 months, 1, 2 at 3 years, with no DP in survivors beyond 6 months(n = 50). Functionally, NKG2C+ NK cells showed a higher production of IFN-gamma in patients without DP at 30 to 90 days. In normal donors, IFN-gamma production was increased several folds on incubation with CTLA4Ig for 24 hours, supporting the anti-leukemia potential of CTLA4Ig-DLI. On multivariate analysis, NKG2C+/ NKG2A- NK cells had the strongest impact on DP (OR-0.3[0.1-0.6]). Likewise, OS strongly correlated with NKG2C+NK cells (p <0.001). Low levels of NKG2C+NK cells at day+60 and +180 correlated with increased acute and chronic GVHD respectively. They reverted to higher and sustained levels after resolution of chronic GVHD. <h3>Conclusions</h3> In patients with advanced leukemia receiving grafts from CMV seropositive donors, rapid surge of functional NKG2C+NK cell subsets, irrespective of CMV reactivation post-HCT, was observed following CTLA4Ig-DLI. CTLA4Ig-DLI probably promotes an early and sustained NKG2C+NK cell surge with functional anti-leukemia potential demonstrated both in-vitro and in-vivo, an absence of which both numerically and functionally, strongly correlated with early DP. Importantly, NKG2C+NK cells remained persistently elevated, even beyond two years post-transplantation and was the predominant factor associated with disease-free survival without GVHD, on long-term follow-up.