CD200R1 Distinguishes Uncommitted Precursors from Functionally Mature NK Cells within the Human Tonsil Stage 4A NK Cell Population

Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) whose development and anti-tumor functions can be critical for the successful treatment and long-term disease-free survival of patients with hematologic malignancies. In humans, NK cells derive from bone marrow resident hematopoietic progenitor cells that traffic to secondary lymphoid tissues (SLTs) where they complete their terminal differentiation and maturation through a series of developmental stages before returning to the blood as mature NK cells. Although major stages of human NK cell development in SLTs have been clearly defined according to the differential surface expression of CD34, CD117, CD94, NKp80, CD16, and CD57 among lineage antigen (Lin) negative lymphocytes, continued investigation has revealed additional phenotypic and functional heterogeneity at each developmental stage. Through extensive ex vivo single-cell RNA sequencing and flow cytometry analyses we have identified two subsets of tonsil-resident Lin ^-CD34 ^-CD117 ^+/-CD94 ⁺NKp80 ^-CD16 ^-CD57 ^- stage 4A NK cells. These two subsets differ in their expression of the inhibitory receptor, CD200R1, which is not expressed by mature NK cells in the peripheral blood from healthy individuals. The majority of stage 4A cells expressed high amounts of surface CD200R1, which correlated with low gene and undetectable protein expression of intracellular cytolytic granules (perforin and granzymes A, B, K, and M), killer immunoglobulin-like receptors (KIRs), and transcription factors required for terminal NK cell maturation (EOMES, T-BET). In addition, upon ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, CD200R1 ⁺ stage 4A NKDIs did not produce interferon-gamma (IFN-g), a hallmark feature of mature NK cells. In contrast, many CD200R1 ^- stage 4A cells constitutively expressed perforin, granzymes, EOMES, and/or T-BET; many expressed KIRs; and many produced IFN-g upon ex vivo stimulation. Furthermore, the frequency of KIR ⁺ cells among CD200R1 ^- stage 4A cells was significantly higher than that among autologous tonsil stage 4B NK cells (Lin ^-CD34 ^-CD117 ^+/-CD94 ⁺NKp80 ⁺CD16 ^-CD57 ^-) (20.8 ± 1.65 vs. 8.12 ± 1.66; p < 0.01; n = 14), suggesting that as a population CD200R1 ^- stage 4A cells are potentially out of sequence in terms of the linear NK cell developmental pathway. Based on these ex vivo findings, we hypothesized that CD200R1 ⁺ stage 4A cells represent NK cell precursors, whereas the CD200R1 ^- stage 4A population contains more mature NK cells that lack NKp80, CD16, and CD57. To further address this hypothesis and to determine their ex vivo potentials for NK cell and non-NK ILC differentiation, we cultured CD200R1 ⁺ and CD200R1 ^- stage 4A cells in vitro in the presence of OP9-DL1 stroma and recombinant human IL-7 and IL-15, conditions previously shown to support all human ILC and NK cell subset differentiation. Under these conditions, both stage 4A populations generated NKp80 ⁺ NK cells in bulk and single-cell clonal assays, whereas neither population gave rise to ILC2s (CD294 ⁺) which precede stage 4A NK cells in the developmental scheme. However, while the majority of cultures derived from CD200R1 ⁺ stage 4A clones contained ILC3s (CD94 ^-NKp44 ⁺), significantly fewer clones from CD200R1 ^- stage 4A cells produced ILC3s (7 of 26 CD200R1 ^- clones vs. 20 of 23 CD200R1 ⁺ clones; p = 0.000587). Moreover, none of the CD200R1 ^- stage 4A-derived clonal cultures that contained KIR ⁺ NK cells contained ILC3s, suggesting that the majority of CD200R1 ^- stage 4A cells are lineage committed NK cells. Collectively, these data further characterize the heterogeneity of the human tonsil stage 4A NK cell population and identify CD200R1 as a marker distinguishing uncommitted precursor cells from a minor population of cells with otherwise mature NK-associated phenotype and function. In light of the role of CD200R1 in regulating lymphocyte functions in the setting of cancer, further research is warranted to determine its potential role(s) in regulating human NK cell development.