Defining Early Human NK Cell Developmental Stages in Primary and Secondary Lymphoid Tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56brightCD16+/− and CD56dimCD16+ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy

Expression of KIR, NKG2A/C, NCR, NKG2D and CD244 within the CD56^dimCD16⁺ NK cell subset of committed NK cells in bone marrow (BM), cord blood (CB), peripheral blood (PB), inguinal LN (inLN), liver LN (liLN) and spleen (SPL) (all n = 5).

<p>(A) Shown are the percentages of CD56^dimCD16⁺ cells positive for each specific receptor within each tissue; *<i>P</i><.05, **<i>P</i><.01. (B) Shown is the mean fluorescence intensity (MFI) for each specific receptor expressed by CD56^dimCD16⁺ cells; *<i>P</i><.05, **<i>P</i><.01.</p

Expression of CD133, CD33, CD244 and NKG2A within the NK cell developmental stages in bone marrow (BM) (n = 5).

<p>Left panels show the percentages of cells positive for the specific markers. Right panels show the mean fluorescence (MFI) of each specific marker.</p

Distribution of the NK cell developmental stages in different human tissues.

<p>Shown are the results for bone marrow (BM), cord blood (CB), peripheral blood (PB), inguinal LN (inLN), liver LN (liLN) and spleen (SPL) (all n = 5). Shown are all NK cell developmental stages within each tissue; *<i>P</i><.05, **<i>P</i><.01, ***<i>P</i><.0001.</p

Developmental stages of NK cells in BM (continued).

<p>Further identification of developmental NK cell stages in BM based on expression of CD133, CD34, CD33, CD177, CD244, NKG2A, CD56 and CD94 using flow cytometry panel 1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030930#pone-0030930-t001" target="_blank">Table 1</a>). Indicated is the presence of each specified marker within each stage (based on the percentage of positive cells present): + = 100–80%; +/−<80%; − = below reliable detection limits.</p

Proposed model for human NK cell development <i>in vivo</i>.

<p>Based on our data, we propose that precursor NK cells (stage 2) may traffic from BM to LN, where commitment to the NK cell lineage may take place (stage 3a→3b) followed by potential <i>in situ</i> differentiation of NK cells with restricted maturation of the NK cell receptor repertoire. For further differentiation of committed NK cells, CD56^dim (stage 3b) or CD56^bright cells (stage 4) may traffic towards splenic tissue in which CD56^dim cells may develop and further maturation of the NK cell receptor repertoire can take place. Final maturation of NK cells may occur through trafficking of cells towards the periphery from which NK cells may be further distributed to different compartments in the human body.</p

Identification of seven NK cell developmental stages in bone marrow (BM).

<p>Based on the stages defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030930#pone-0030930-t001" target="_blank">Table 1</a>, we analyzed the presence of the different NK cell developmental stages in BM. Shown is one representative example (n = 5). Cells were gated on the CD45⁺CD3⁻ population within CD45⁺/SS gated cells to exclude T cells and endothelial cells from analysis. Subsequently, cell subsets were divided based on the expression of CD34 and CD117. From there, each subset was analyzed for CD56 and CD94 expression, leading to the identification of seven NK cell developmental stages: 1, 2, 3a, 3b, 4, 5a, 5b.</p