Phenotypic and Functional Characteristics of NK Cells Associated with CMV Infection after Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

Cytomegalovirus (CMV) infection represents a major complication in hematopoietic stem cell transplantation (HCT). There is accumulating evidence that immune responses to CMV infection involve the expansion of specific subsets of NK cells largely driven by the activating receptor NKG2C. We prospectively examined the reconstitution of NK cells post-HCT (days 21, 30, 45, 80 and 120) for their immunophenotypes and functions. Of the total 111 patients enrolled, the current analyses focus on 82 CMV seropositive recipients with complete and longitudinal immunologic data available (median age: 55, range 19-70) who received HCT (PBSC: n=75, BM: n=4, UCB: n=3) from related (n=36) or unrelated donors (n=46) after fully ablative (n=22) or reduced-intensity (n=60) conditioning. Donor CMV serostatus was positive in 56, negative in 26. CMV reactivations occurred in 19 patients (23.2%). % of NK cells expressing the following markers/cytokines were evaluated in multivariate analyses: NKG2C, granzyme B (GrB), Ki67, CD107, CD137, IFNg, and PD1. P-values are unadjusted unless specified otherwise. To evaluate multiple test parameters, we used “q-value” which is a false discovery rate incurred when calling the finding significant. Consistent with earlier reports, CMV reactivation was significantly associated with increased %NK cells expressing NKG2C (6.1%, p=0.034, q=0.017) as well as GrB (25.5% increase, p=0.002, q<0.001) and Ki67 (5.6%, p=0.004, q<0.01). When we examined characteristics of NKG2C+NK cells in samples on day +80, activation markers, CD107, IFNg, CD137, and Ki67, were significantly over-expressed compared with NKG2C-NK cells (by 15.2%, [p<0.001], 11.0% [p<0.001], 5.0% [p<0.001], 1.7% [p=0.002], respectively) while GrB expression was lower (-8.4%, p=0.025) (adjusted p). We further explored possible relationship between in vitro T cell responses to CMVpp65 Ag and NK cell phenotypes. In day +80 samples, % CMVpp65-induced IFNg+CD3+T cells significantly associated with NK cells expressing NKG2C (p=0.007, q<0.001), suggesting a coordinated response to CMV between innate and adaptive immune responses. CMVpp65-specific IFNg+CD3+ T cells were also associated with IFNg+ response in the NKG2C- population (p<0.001, q<0.001) while the association was less significant in the NKG2C+ population (p=0.091, q=0.029). Similarly, pp65-reactive IFNg+CD3+ T cells associated with CD137 expression in NKG2C-NK cells (p=0.007, q=0.003) but not in NKG2C+NK cells (p=0.8, q=0.19). In summary, our data support that CMV reactivation is associated with expansion (Ki67) and cytotoxic functions (GrB, IFNg) of NK cells expressing NKG2C following HCT. The data also suggest that there are coordinated immune responses between T cells and NK cells with NKG2C- population showing a greater reactivity with in-vitro CMVAg stimulation.