Abstract

Natural killer (NK) cells are the first subset of lymphocytes to reconstitute, and likely play an important role in the graft-versus-leukemia response early after allogeneic hematopoietic stem cell transplantation. NK cells are highly diverse, and express an array of activating and inhibiting receptors, which delicately regulate their activation status.

To provide a framework to better understand NK cell development and maturation after cord blood transplantation (CBT) and its implication on clinical outcomes, we designed a mass cytometry (also known as cytometry by time of flight) panel of 40 monoclonal antibodies recognizing lineage markers, NK cell receptors and transcription factors to interrogate the NK cell repertoire in prospectively collected peripheral blood (PB) samples from 60 CBT recipients. The resultant high-dimensional single cell data were analyzed by t-Distributed Stochastic Neighbor Embedding (t-SNE) dimension reduction algorithm to generate expression profile graphs.

With this mass cytometry platform, we first interrogated and compared the NK cell repertoire in CB (n=10) and adult peripheral blood (PB) (n=20) and used inverse Simpson index to measure the diversity of the NK cell populations. NK cells from CB donors had a significantly lower diversity (median 537, range 311-891) compared to NK cells from healthy adult PB donors (median 2858, range 1228-5630; p<0.0001). Since CMV infection has been shown to drive NK cell maturation, we next asked if CMV infection also alters NK cell diversity by comparing the diversity of the NK cell repertoire in CB (n=10), and in PB from adult healthy donors who were either CMV-seropositive (n=10), or CMV-seronegative (n=10). The diversity of the NK cell repertoire was significantly greater in CMV seropositive healthy individuals (median 4217, range 2672-5630) compared to CMV-seronegative healthy adults (median 2247 , range 2026-2468; P=0.0002) and cord blood donors (median 537, range 311-891; P<0.0001), pointing to an important role for CMV infection in driving NK diversity.

Harnessing mass cytometry, we undertook an in-depth examination of the NK repertoire in prospectively collected PB samples from 60 CBT recipients. We first sought to determine if CMV infection early after CBT can drive NK cell maturation and accelerate the acquisition of a diverse repertoire. The median time to CMV reactivation for the cohort was 71 days. CMV reactivation in the first 100 days after CBT (n=25) was associated with significantly greater NK diversity (median 844, range 198-1196) when compared to patients without CMV reactivation (median 405, range 110-889; p=0.0003). Notably, CMV augmented NK diversity mainly in the KIR+ population (median 743, range 207-1500) compared to the KIR- population (median 361, range 92-1038; p=0.007); while in the absence of CMV reactivation, NK cell diversity in the KIR+ and KIR- populations remained similar (median 305, range 102-702 vs. median 332, range 54-830; p=0.98). These data indicate that CMV infection augments NK diversity after CBT, and that this effect is most pronounced within the KIR+ population.

We next determined if NK cell diversity can influence outcomes by comparing the diversity of the NK cell repertoire in PB samples collected in the first 100 days after CBT from 15 patients who went on to relapse compared to 45 patients who remained in remission for 2 years. NK cell diversity was significantly higher (median 839, range 416-1069) in patients who remained in remission compared to those who progressed and relapsed (median 446, range 328-925; p=0.03). Moreover, the greatest difference in diversity in the two groups was noted within the KIR+ population (median 788, range 101-1500 for patients in remission vs median 320, range 177-857 for patients who relapsed; p=0.002).

In summary, we have used multiparametric mass cytometry to examine expression of 40 NK markers, and to gain insights into the diversity of the NK cell repertoire after CBT. We have shown that CB NK cells have a limited NK cell diversity, which increases in breadth and subsets after transplant. Moreover, we observed that CMV infection reconfigures the NK cell compartment and drives the diversity of the NK cell repertoire, especially within the KIR+ population. These data may provide the basis to rationally design therapeutic strategies that can modulate NK diversity in the settings of infection and transplant.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.