Clonal Expansion and Long-Term Persistence of Rhesus Macaque NK Cells with an Adaptive Phenotype

Natural killer (NK) cells are defined as lymphocytes capable of target cell killing and cytokine production independent of somatic antigen receptor recombination. Challenging the paradigm of continuous production of non-specific cytotoxic NK cells from undifferentiated precursors, a subset of murine NK cells sharing many properties with cytotoxic effector T cells was shown to specifically respond to a viral infection, resulting in expansion and protective immunity upon adoptive transfer, uncovering unexpected "adaptive" capabilities for NK cells. In humans, CD56^dimCD16⁺ NK cells expressing NKG2C and variegated patterns of KIR are associated with Cytomegalovirus (CMV) infection, and may represent human analogs of murine adaptive NK cells. In contrast to CD56^bright and canonical CD56^dim NK cells, we recently showed that human adaptive NK cells lack expression of specific signaling proteins and transcription factors, and epigenetically resemble CD8⁺ effector T cells, suggesting shared pathogen-driven developmental pathways (Schulms et al, 2015). NK cell heterogeneity, ontogeny, and mechanisms sustaining NK cell memory are poorly understood, particularly in humans, because in contrast to adaptive T and B cells, clonal tracking of NK cells based on receptor gene structure is not possible. We applied autologous transplantation of genetically barcoded CD34+ rhesus macaque hematopoietic stem and progenitor cells (HSPC) to interrogate the landscape of NK cell production and expansion at a clonal level. We followed 3 monkeys for up to 4 years post-transplantation, and observed a distinct clonal ontogeny for CD56^-CD16⁺ (corresponding to human CD56^dimCD16⁺ NK) versus CD56⁺CD16^- rhesus NK cells. CD56^-CD16⁺ NK were oligoclonal, and dominated by small numbers of highly biased clones, a pattern showing little overlap with highly polyclonal multipotent contributions to T, B, myeloid and CD56⁺CD16^- NK cell lineages. There were clear expansions and contractions of groups of these highly biased CD56^-CD16⁺ NK cell clones over time, with individual clones contributing up to 20% to this lineage, similar to patterns of waxing and waning T cell clones, suggesting responses of both cell types to extrinsic cues such as viruses. In contrast, all other lineages, including CD56⁺CD16^- NK cells, were characterized by very stable contributions from long-term engrafting multipotent HSPC clones over time. The highly biased, greatly expanded, and waxing and waning CD56^-CD16⁺ NK cell clones were specifically enriched in a subpopulation of CD56⁺CD16^- NK with high KIR and low SYK expression,and decreased methylation at IFNG the CNS1 locus, analogous to human CMV-associated adaptive NK cells. This population of cells was increased in CMV+ compared to CMV- macaques; our transplanted macaques were CMV+. Canonical CD56⁺CD16^- NK cells, lacking an adaptive phenotype, had clonal patterns similar to CD56⁺CD16^- NK cells. We profoundly depleted CD56^-CD16⁺ NK cells with an anti-CD16 antibody, and observed rapid regeneration of the same CD56^-CD16⁺ NK clonal pattern, without recruitment of contributions from multipotent HSPC via CD56⁺CD16^- putative precursor NK cells. There was a marked increase in cycling of CD56^-CD16^dim NK cells just prior to reappearance of CD56^-CD16^bright NK cells, and no change in cycling of CD56⁺CD16^- cells. Thus, adaptive NK cell clones, both at steady state and under regenerative stress do not appear to require ongoing production from HSPC via CD56⁺CD16^- precursors. These results suggest that adaptive NK cells or their immediate precursors have acquired self-renewal properties and high proliferative potential, with clonal restriction potentially based selection and expansion of cells with specific, epigenetically-controlled receptor expression able to interact with external stimuli such as viruses. In conclusion, via tracking of individual NK cell clonal histories in rhesus macaques, we provide the first direct evidence for persistence of adaptive NK cell clones in any species, offering potential insights into the acquisition of NK cell memory.