Natural Killer Cell Dysfunction in Chronic Lymphocytic Leukemia Is Associated with Loss of the Mature KIR3DL1⁺ Subset

Background: B-cell chronic lymphocytic leukemia (CLL) is a common blood cancer characterized by high prevalence of malignant B cells in peripheral blood. Small lymphocytic lymphoma (SLL) is considered to be a different presentation of the same disease, with the malignant B cells primarily localized in lymph nodes. Natural killer (NK) cells are innate immune effectors that can spontaneously identify and kill malignant cells, especially hematopoietic cancers. In peripheral blood of CLL patients, NK cells are chronically exposed to significant tumor burden, which is predicted to influence their phenotype and function. Effective NK cell function may be particularly beneficial in CLL patients, since commonly-used monoclonal antibody therapies (e.g. rituximab, alemtuzumab) rely at least partially on ADCC-mediated by NK cells.

Methods: We performed a prospective analysis of biomarkers on fresh peripheral blood lymphocytes from 25 untreated CLL patients, 10 untreated SLL and 17 age-matched healthy controls by 10-color flow cytometry. All subjects signed IRB approved informed consent forms. Our study analyzed 180 distinct biomarker parameters, with a particular focus on NK and T cells. Differences in biomarker expression between patients with SLL, CLL, and healthy controls were compared by Wilcoxon rank-sum test.

Results: Absolute numbers of NK and T cells per µl of blood were significantly higher in CLL patients, and this correlated with increased B cell numbers. As indicators of immune suppression, the frequency of regulatory T cells was significantly increased in CLL samples, as were levels of PD-1 expression on T cells and CD56^dim NK cells. NK cells in CLL expressed higher levels of CD27, which is characteristic of a less mature phenotype, and CD56^dim cells expressed lower levels of NKG2D. Compared to healthy controls, CLL samples displayed a marked reduction in degranulation by CD56^dim NK cells in response to transformed 721.221 B cells, either with or without rituximab. CD56^dim NK cells from CLL patients were also less viable under resting conditions or when challenged with target cells, especially in ADCC responses. We further observed a striking reduction in the frequency and viability of KIR3DL1⁺ NK cells, which progressed over time in most CLL patients. Surprisingly, CLL patients with the highest levels of PD-1 expression on NK cells possessed genes for both KIR3DL1 and its ligand, HLA-Bw4. Our findings were also clearly evident in a CLL patient compared to her healthy monozygotic twin, thereby providing compelling support for the results in the full patient cohort. The altered expression levels of nearly all of the NK cell biomarkers and degranulation were less pronounced in blood samples from SLL patients, presumably due to low tumor burden in peripheral blood.

Conclusions: CLL patients have increased numbers of NK cells in peripheral blood, but these NK cells are less mature, are significantly depleted of the KIR3DL1⁺ subset, and have deficits in degranulation response, reduced expression of NKG2D activating receptor, increased expression of inhibitory PD-1, and enhanced susceptibility to activation-induced death when challenged with tumor targets and rituxumab. Our findings support the hypothesis that immune dysfunction in CLL may be due in part to a selective loss of mature KIR3DL1⁺ NK cells, possibly upon encountering overwhelming tumor burden in peripheral blood, and CLL patients may benefit from therapeutic strategies that augment NK cell function.