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موجز عن البحث:
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Cord blood (CB) is increasingly used as a
source of stem cells for hematopoietic stem cell transplantation, and natural
killer (NK) cells may be the effectors of the antileukemic response observed
after CB transplantation. Here, we analyzed the phenotype and functions of CB
NK cell subsets. We determined that the percentage of NK cells was higher in
CB compared with peripheral blood (PB). Furthermore, there was a higher
percentage of the CD56(bright) subset in CB. CB NK cells reached a late stage
of differentiation, but exhibited higher expression of NKG2A and expressed
fewer killer-cell immunoglobulin-like receptors, suggesting an incomplete
maturation. CB NK cells highly expressed CXCR4, but did not express
L-selectin, highlighting unique homing properties of CB NK cells. CB NK cells
proliferated in response to interleukin-2 and degranulated in response to
stimulation with tumor cells, but failed to lyse K562 cells in (51)Cr-release
assay. CB NK cells exhibited a lower interferon-γ production in comparison
with PB NK cells. Culture with IL-2 increased CB NK cell functions. Our study
sheds light on CB NK cell properties and highlights the potential of CB as a
source of NK cells for immunotherapy.
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ملخص المشاركة:
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Cord
blood (CB) has recently been used as an alternative source of haematopoietic
stem cells (HSCs) for transplantation, as it has clinical advantages over
other sources of HSCs including: off the shelf availability, less stringent
human leucocyte antigen matching, reduced incidence and severity of graft-versus-host disease (GvHD), and preservation
of the graft- versus -leukaemia
(GvL) effect. Natural Killer (NK) cells are prominent in CB constituting up
to 30% of the total mononuclear cells, and their reconstitution occurs early
after transplantation. Hence, NK cells are key effectors in mediating the GvL
effect in CB transplantation settings. In this study we aimed to investigate
whether CB NK cells can be activated by cytokines and become fully functional
as for peripheral blood NK cells. Cytokines involved in stimulation and
proliferation of NK cells were used including Interleukin (IL)-2, IL-12,
IL-15, IL-18, the combination of IL-15 and IL-18 or the combination of IL-15
and IL-2. We studied both the phenotypic characteristics and functional
capabilities of cytokine-activated CB NK cells. A thorough investigation and
comparative study to peripheral blood NK cells was carried out in each
experiment. We investigated expression of cytokine receptor, the phenotype of
activated CB NK cells, capability of NK cells to proliferate in the presence
of cytokines, signalling pathway for IL-2 and IL-12, IFN-g and TNF-a
production, and killing capacity. Our results indicated that CB NK cells are
readily responsive to cytokine treatment presenting a comparative amount of
cytokines’ receptors. However, CB NK cells express significantly less
receptors of IL-2 explaining less activation acquired by IL-2. CB NK cells
acquire the phenotype of activated NK cells upon activation with cytokines
showing up-regulation of activation markers, adhesion molecules, and secondary
lymphoid tissues homing repertoire. Testing CB NK cell cultures’ supernatants
revealed high production of IFN-g but not TNF-a. Nonetheless, differential
findings depending on which cytokine used was observed. CB NK cells gain the
tendency to proliferate in the presence of cytokines diluting the CFSE
fluorescence by day 2. Interestingly, better proliferation was observed in
cultures containing a combination of IL-15 and IL-18. Cytokines’ treated CB
NK cells when co-cultured with K562 were able to kill this cell line in 51Cr
release cytotoxicity assay. All together, this study aims to identify which
cytokine or a combination of cytokines promotes fully functional CB NK cells
that could be used for therapeutic purposes.
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ملخص المشاركة:
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Natural killer (NK) cells play substantial
role in clearing infected cells and tumour cells, and in mediating graft
versus leukaemia effect (GvL) in transplantation settings. In this study, we
aimed to investigate the mechanisms of cord blood (CB) NK cell activation and
compare it to peripheral blood (PB) NK cells to identify which conditions
lead to fully activated functional NK cells for immunotherapy. Cytokines
involved in stimulation and proliferation of NK cells were used including
Interleukin (IL)-2, IL-12, IL-15, IL-18, thecombination of IL-15 and IL-18 or
the combination of IL-15 and IL-2. We studied both the phenotypic
characteristics and functional capabilities of cytokine-activated NK cells.
Our results indicated that CB NK cells are readily responsive to cytokine
treatment presenting a comparative amount of most cytokines’ receptors.
However, CB NK cells express significantly less IL-2 receptors correlating
with the lower levels of activation reached after IL-2 stimulation. CB NK
cells acquire the phenotype of activated NK cells upon ctivation with
cytokines showing up-regulation of activation markers, adhesion molecules,
and secondary lymphoid tissues homing markers. The analysis of CB NK cell
cultures’ supernatants revealed high production of IFN-g and inflammatory
cytokines (IL-6, IL-8) but only traces of TNF-a. Nonetheless, differential
findings depending on which cytokine used was observed. CB NK cells gained
the tendency to proliferate in the presence of cytokines diluting CFSE by day
2. Interestingly, better proliferation was observed in cultures containing a
combination of IL-15 and IL-18. Cytokines’ treated CB NK cells when
co-cultured with K562 were able to kill this cell line in 51Cr release
cytotoxicity assay. Our early finding of relative gene expression revealed
significant differences in FN-g, Granzyme-B, Perforin, and BCL-2 expression
based on the cytokine condition used. Our results from transwell migration
assay revealed that CB NK cells when treated with the combination of IL-15
and IL-2 or IL-15 and IL-18 were able to migrate toward CCL19/21 and
CXCL10/11 chemokine signal showing similar pattern of migration to PB NK
cells. Using cytokines, it was also feasible to induce memory NK cells from
CB NK cells that exhibited sustained ability to produce IFN-g at day 21. All
together, this study aims to identify which cytokine or combination of
cytokines promotes fully functional CB NK cells that could be used for
therapeutic purposes.
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