CD56dimCD57+NKG2C+ NK cell expansion is associated with reduced leukemia relapse after reduced intensity HCT

Abstract

We have recently described a specialized subset of human natural killer (NK) cells with a CD56(dim)CD57(+)NKG2C(+) phenotype that expand specifically in response to cytomegalovirus (CMV) reactivation in hematopoietic cell transplant (HCT) recipients and exhibit properties characteristic of adaptive immunity. We hypothesize that these cells mediate relapse protection and improve post-HCT outcomes. In 674 allogeneic HCT recipients, we found that those who reactivated CMV had lower leukemia relapse (26% (17-35%), P=0.05) and superior disease-free survival (DFS) (55% (45-65%) P=0.04) 1 year after reduced intensity conditioning (RIC) compared with CMV seronegative recipients who experienced higher relapse rates (35% (27-43%)) and lower DFS (46% (38-54%)). This protective effect was independent of age and graft-vs-host disease and was not observed in recipients who received myeloablative regimens. Analysis of the reconstituting NK cells demonstrated that CMV reactivation is associated with both higher frequencies and greater absolute numbers of CD56(dim)CD57(+)NKG2C(+) NK cells, particularly after RIC HCT. Furthermore, expansion of these cells at 6 months posttransplant independently trended toward a lower 2-year relapse risk. Together, our data suggest that the protective effect of CMV reactivation on posttransplant relapse is in part driven by adaptive NK cell responses.

Figure 1. CMV reactivation is associated with reduced relapse risk and superior disease-free survival in RIC, but not MA HCT recipients

Kaplan-Meier curves of (A) relapse rates and (B) DFS stratified by CMV status in RIC recipients. (C) Relapse rates and (D) DFS stratified by CMV status in MA recipients. Blue dashed lines represent trends calculated for CMV seronegative recipients. Green dotted lines represent trends calculated for CMV seropositive recipients that did not experience viral reactivation. Red solid lines represent trends calculated for CMV seropositive recipients that experienced viral reactivation. p values shown in each plot were calculated for trends.

Figure 2. Preferential expansion of CD56^dimCD57⁺NKG2C⁺ adaptive NK cells in RIC HCT recipients that experience CMV reactivation

Average percentage (A) and (B) absolute number (cells/μl of blood) of CD56⁺ NK cells with an adaptive CD56^dimCD57⁺NKG2C⁺ phenotype in CMV seronegative recipients at day 100 (RIC n=44, MA n=32), 6 months (RIC n=35, MA n=23), and 1 year (RIC=31, MA=21) post-transplant. Values for CMV seropositive recipients without CMV reactivation at day 100 (RIC n=22, MA n=12), 6 months (RIC=13, MA=14) and 1 year (RIC=11, MA=8) post-transplant are shown in the middle panels. Values for CMV seropositive recipients that reactivated CMV at the time of viral diagnosis (RIC n=28, MA n=18), 2 weeks post-diagnosis (RIC n=26, MA n=14), 4 weeks post-diagnosis (RIC n=29, MA=23), 8 weeks post-diagnosis (RIC n=24, MA n=15), 6 months post-transplant (RIC n=29, MA n=17) and 1 year post transplant (RIC n=26, MA n=10) are shown in the right panels. *p ≤ 0.05 comparing RIC to MA. Error bars represent SEM.

Figure 3. Absolute monocyte counts at the time of CMV reactivation are associated with CD56^dimCD57⁺NKG2C⁺ NK cell expansion

Absolute monocyte counts from 28 CMV seropositive recipients at the time of viral reactivation were plotted against either (A) the absolute number or (B) the percentage of CD56^dimCD57⁺NKG2C⁺ NK cells in peripheral blood samples from these recipients at either 6 months or 1 year. Absolute lymphocyte counts at the time of viral diagnosis from the same recipients were also plotted against either (C) the absolute number or (D) the percentage of CD56^dimCD57⁺NKG2C⁺ NK cells in peripheral blood samples at either 6 months or 1 year.

Figure 4. CD56^dimCD57⁺NKG2C⁺ NK cells produce TNF and IFN-γ at high frequencies relative to other NK cell subsets

PBMCs from CMV seropositive donors were cultured with or without K562 target cells at a 2:1 ratio, and functional responses were analyzed in subsets of CD56^dim NK cells. (A) Histograms of TNF expression (open black lines) and intracellular IFN-γ expression (open black lines) in NK cells cultured with K562 targets compared to effector cells cultured alone (shaded grey lines) for a representative donor. (B) Cumulative TNF and IFN-γ expression data in NK cells cultured with K562 targets from 5 donors. Two independent experiments were performed. * =p≤ 0.05, ** =p≤ 0.005. Two-sided, paired t-tests were used to determine significance. Error bars represent SEM.