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

Background & MethodsCTLA4Ig-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.ResultsAll 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.ConclusionsIn 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. 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. 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. 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.