Early Differentiation of Human CD11c+NK Cells with γδ T Cell Activation Properties Is Promoted by Dialyzable Leukocyte Extracts

Abstract

Reconstitution of the hematopoietic system during immune responses and immunological and neoplastic diseases or upon transplantation depends on the emergent differentiation of hematopoietic stem/progenitor cells within the bone marrow. Although in the last decade the use of dialyzable leukocyte extracts (DLE) as supportive therapy in both infectious and malignant settings has increased, its activity on the earliest stages of human hematopoietic development remains poorly understood. Here, we have examined the ability of DLE to promote replenishment of functional lymphoid lineages from CD34+ cells. Our findings suggest that DLE increases their differentiation toward a conspicuous CD56+CD16+CD11c+ NK-like cell population endowed with properties such as IFNy production, tumor cell cytotoxicity, and the capability of inducing γδ T lymphocyte proliferation. Of note, long-term coculture controlled systems showed the bystander effect of DLE-stromal cells by providing NK progenitors with signals to overproduce this cell subset. Thus, by direct effect on progenitor cells and through activation and remodeling of the supporting hematopoietic microenvironment, DLE may contribute a robust innate immune response by promoting the emerging lymphopoiesis of functional CD11c+ NK cells in a partially TLR-related manner. Unraveling the identity and mechanisms of the involved DLE components may be fundamental to advance the NK cell-based therapy field.

Figures

Figure 1

Dialyzable leukocyte extracts (DLE) promote proliferation of primitive hematopoietic CD34+ cells from neonatal and adult sources. CD34+ cells from adult bone marrow (ABM) and umbilical cord blood (UCB) were enriched and cultured for 72 hours with DLE Transferon and BrdU. Cells were stained for the identification of CD34+ cell progenitors followed by intracellular staining of BrdU. Multiparametric flow cytometry was performed in a FACSCanto II and the cell frequencies of BrdU+ cells calculated. Data are representative of 3 independent experiments. ∗P < 0.05; ∗∗P < 0.01.

Figure 2

CD56+CD11c+ NK-like cells expeditiously develop from early lymphoid progenitors upon DLE stimulation. Upon 24 hours of DLE Transferon stimulation, the whole fraction of CD34+ cells (a and b) or purified primitive cells, including hematopoietic stem cells (HSC), multipotent progenitors (MPP), and early lymphoid progenitors (ELP) (a), were cocultured with MS-5 stromal cells for 30 days under lymphoid lineage conditions. CD56+CD11c+ NK-like cell production was investigated by flow cytometry and reported as cell frequencies (a) or yield per input progenitor (b and c). For comparison, unstimulated cultures were considered as control conditions. DLE, dialyzable leukocyte extracts Transferon. ∗∗P < 0.01.

Figure 3

Stromal cell activation by DLE increases the emergent innate immune cell differentiation. MS-5 stromal cells were exposed or not to DLE Transferon 24 hours before coculturing with CD34+ hematopoietic stem and progenitor cells (HSPC) that were pretreated 24 hours with DLE Transferon (a). All conditions were placed in lymphoid lineage cocultures for 30 days followed by flow cytometry analyses of CD56+ NK cells and CD11c+ DC production. Yields per input progenitor were tabulated to show the positive contribution of activated stromal cells to innate differentiation (b). DLE, dialyzable leukocyte extracts Transferon. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Figure 4

Preferential production of functional CD11c+CD16+ NK-like cells promoted by hematopoietic-stromal cell communication responding to DLE. MS-5 stromal cells were exposed or not to DLE Transferon 24 hours before coculturing with DLE Transferon-pretreated CD34+ hematopoietic stem and progenitor cells (HSPC), as described in Figure 3(a). All conditions were placed in lymphoid lineage cocultures for 30 days followed by flow cytometry analyses, where the indicated gates were used to discriminate CD56+CD11c+ and CD56+CD16+CD11c+NK cell frequencies (a). Yields per input progenitor were tabulated to record the significant variations by stromal or hematopoietic cells exposure to DLE Transferon (b). ∗∗∗P < 0.001.

Figure 5

Effector functions of CD11c+ NK cells differentiated from DLE-induced lymphoid progenitor cells include tumor cytotoxicity and IFN-gamma production. NK cytotoxicity function was evaluated on CFSE+ K562 tumor cells as target of NK-like cells derived from DLE stimulation (DLE-NK) (a). We used 7-aminoactinomycin D (7-AAD) to evidence the frequency of CFSE+7-AAD+ killed target cells. Production of IFN-gamma by CD11c−CD56+ and CD11c+CD56+ NK cells was detected by intracellular staining (b).

Figure 6

Activation of γδ T cells as a result of DLE-associated CD11c+ NK cell emergent production. γδ T cells from healthy donors were cocultured with the DLE-derived CD11c+ NK cell at a ratio of 2 : 1 CD56+CD11c+ NK : γδ T cell (a). Both NK and γδ T cell populations were highly purified by flow cytometry sorting before setting the proliferation assay (b). γδ T lymphocyte proliferation was assessed at 72 hours by dilution of Cell Trace Violet (CTV) dye (c).

Figure 7

Emergent CD11c+ NK cell production contributing the adjuvant immune-surveillance effects of DLE Transferon: a proposed model. Dialyzable leukocyte extracts Transferon promote the early differentiation of functional CD11c+ NK cells endowed with the capabilities of tumor cell cytotoxicity, IFNy production, and γδ T lymphocyte proliferation induction, which may in turn contribute to innate immune responses against virus-infected or tumor cells. The potential involved mechanisms require further investigation.