Stem Cell Reports

Article

Improved hematopoietic stem cell transplantation upon inhibition of natural

killer cell-derived interferon-gamma
Lorena Lobo de Figueiredo-Pontes,1,13 Miroslava K. Adamcova,2,3,13 Srdjan Grusanovic,2,3,4
Maria Kuzmina,2,4 Izabela Aparecida Lopes,1 Amanda Fernandes de Oliveira Costa,1 Hong Zhang,5
Hynek Strnad,6 Sanghoon Lee,7 Alena Moudra,8 Anna T. Jonasova,8 Michal Zidka,9,10 Robert S. Welner,5,11
Daniel G. Tenen,5,12,14,* and Meritxell Alberich-Jorda2,3,14,*
1Hematology Division, Department of Medical Images, Hematology, and Clinical Oncology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão

Preto, SP 14048-900, Brazil

2Department of Hemato-oncology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague 142 00, Czech Republic
3Childhood Leukaemia Investigation Prague, Department of Pediatric Haematology and Oncology, 2nd Faculty of Medicine, Charles University in Prague,

University Hospital Motol, Prague 150 06, Czech Republic

4Faculty of Science, Charles University, Prague 128 00, Czech Republic
5Harvard Stem Cell Institute, Harvard Medical School, Boston, MA 02115, USA
6Department of Genomics and Bioinformatics, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague 142 00, Czech Republic
7Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
81st Department of Medicine - Department of Haematology, First Faculty of Medicine, Charles University in Prague and General University Hospital, Prague

120 00, Czech Republic

9Orthopaedic Department CLPA-Mediterra, Prague 190 00, Czech Republic
103rd Medical Faculty, Charles University, Prague 100 00, Czech Republic
11Department of Medicine, Division Hematology/Oncology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
12Cancer Science Institute, National University of Singapore, Singapore 117599, Singapore
13These authors contributed equally
14Co-senior author

*Correspondence: [email protected] (D.G.T.), [email protected] (M.A.-J.)

https://doi.org/10.1016/j.stemcr.2021.06.008

SUMMARY

Hematopoietic stem cell transplantation (HSCT) is a frequent therapeutic approach to restore hematopoiesis in patients with hematolog-
ic diseases. Patients receive a hematopoietic stem cell (HSC)-enriched donor cell infusion also containing immune cells, which may have
a beneficial effect by eliminating residual neoplastic cells. However, the effect that donor innate immune cells may have on the donor
HSCs has not been deeply explored. Here, we evaluate the influence of donor natural killer (NK) cells on HSC fate, concluded that NK
cells negatively affect HSC frequency and function, and identified interferon-gamma (IFNg) as a potential mediator. Interestingly,
improved HSC fitness was achieved by NK cell depletion from murine and human donor infusions or by blocking IFNg activity.
Thus, our data suggest that suppression of inflammatory signals generated by donor innate immune cells can enhance engraftment
and hematopoietic reconstitution during HSCT, which is particularly critical when limited HSC numbers are available and the risk of
engraftment failure is high.

INTRODUCTION graft can also affect donor HSC properties and modify
engraftment.
Hematopoietic stem cell transplantation (HSCT) is the only Recent evidence has elucidated that hematopoietic cells,
curative therapy for many hematologic diseases where he- particularly T lymphocytes, known for their ability to
matopoiesis needs to be replaced by healthy hematopoietic trigger inflammatory responses, may directly or indirectly
cells. The success of HSCT mostly depends on the speed affect the function of hematopoietic stem and progenitor
and quality of the hematopoietic recovery in the early cells (Geerman and Nolte, 2017). Hirata and colleagues
post-transplantation phase. Although the main goal of showed that bone marrow (BM) cluster of differentiation
the procedure is to inject hematopoietic stem cells (HSCs) (CD) 4 memory T cells and memory regulatory T cells
with high self-renewal potential to fully reestablish hema- (Tregs) coordinate each other to generate extracellular
topoiesis, blood- or bone marrow-derived donor stem cell adenosine via CD39 and CD73, maintaining HSC quies-
sources also contain committed progenitors and immune cence (Hirata et al., 2018). Similarly, memory CD8+ T cells
cells. Presence of immune cells in the donor sample has support the maintenance of HSCs in the BM (Geerman
been associated with prevention of infections (Chen et al., 2018). Nevertheless, in a transplantation setting us-
et al., 2006; Storek et al., 1997; Tomblyn et al., 2010) and ing a xenotransplant model, it was demonstrated that
graft-versus-tumor effects (Venstrom et al., 2010; Wagner donor-derived activated memory T cells present in unfrac-
et al., 2005). However, besides these well-known effects, tionated umbilical cord blood (UCB) were associated with
it may be hypothesized that immune cells from the reduced HSC engraftment in comparison with CD34+

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 j ª 2021 The Authors. 1999
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
fractionated UCB. This effect was attributed to tumor ne- plantation. By using CCAAT-enhancer-binding protein
crosis factor alpha (TNFa) cytokine produced by these gamma (Cebpg) conditional knockout (KO) mice, we re-
memory T cells and was reverted by TNFa receptor inhibi- vealed that the effect is mediated by the production of cy-
tion (Wang et al., 2017). On the contrary, several studies tokines such as IFNg. Importantly, addition of IFNg-block-
showed that, when HSCs are limiting, memory CD8+ ing antibodies to HSC-NK co-cultures prevented the NK
T cells have a beneficial effect on HSPC engraftment, detrimental effect on HSC, and administration of IFNg-
both in allogeneic and autologous transplantation (Russell blocking antibodies during the early phase post transplant
et al., 2015; Touzot et al., 2015; Triplett et al., 2015). Alto- enhanced human HSC engraftment and hematopoietic
gether, these observations highlight the relevance of the reconstitution. Altogether, our study demonstrates that
immune cells present in the graft, and question their pres- donor NK cells present in the graft can negatively affect
ence as an advantage or disadvantage in a transplantation the early post-transplantation phase, and that modulating
setting. inflammatory signals such as IFNg can contribute to
Natural killer (NK) cells are lineage-specific lymphocytes improved engraftment during HSCT.
with effector functions of cytotoxicity and cytokine pro-
duction. NK cell activity depends on a dynamic balance be-
tween the expression of activating receptors (which recog- RESULTS
nize stress-induced ligands on infected or tumor cells),
inhibitory receptors (which predominantly bind major his- NK cells reduce HSC maintenance and function in
tocompatibility complex [MHC] class I molecules), and culture
cytokine receptors (Kim et al., 2005; Vivier et al., 2008). To investigate potential HSC alterations when exposed to
Upon stimulation, NK cells are also able to produce cyto- NK cells, we developed a co-culture system of sorted mu-
kines such as interleukin (IL)-1, IL-3, IL-4, IL-5, IL-6, granu- rine HSCs and NK cells (Figure 1A). HSCs were defined by
locyte colony-stimulating factor (G-CSF), granulocyte- cell surface markers as lineage negative (Lin), Sca-1+,
macrophage colony-stimulating factor (GM-CSF), macro- c-kit+, CD48, CD150+, and NK cells as Lin, CD3, and
phage colony-stimulating factor (M-CSF), TNFa, and NK1.1+. Co-cultures were established on an OP9 stromal
IFNg, which can potentially influence the properties of cell layer with appropriate HSC culture conditions in the
self-renewal, apoptosis, mobilization, and differentiation presence of the NK cell activating cytokine IL-2 (Figure 1A).
of HSCs and determine different outcomes to hematopoie- Using fluorescence-activated cell sorting (FACS) analysis,
sis (Baldridge et al., 2010; Buza-Vidas et al., 2006; Schuett- we observed a reduction of phenotypically defined HSCs af-
pelz and Link, 2013; Zhang and Lodish, 2008). However, ter 4 days of culture when NK cells were present. This reduc-
besides its cytotoxic effects mediated by HLA missense tion was dependent on the NK/HSC ratio, and was more
recognition signals in HSCT, a potential role in the regula- profound as the number of NK cells in culture increased
tion of HSC functions in transplantation has not been (Figures 1B and S1). Next, we performed colony culture as-
explored. says to assess whether the presence of NK cells would
In the transplant setting, the immune content of the compromise HSC function in vitro (Figure 1C). Colony-
graft may be activated or modified by the pre-transplant forming units were enumerated at day 10 of culture, and
conditioning regimen based on chemotherapy and/or we observed a significant reduction in the presence of NK
radiotherapy (Joncker et al., 2010; Mehta et al., 2015). cells. Similar to our previous result, the number of colonies
Both T and NK cells can recognize and kill residual tumor was reduced in a dose-dependent manner according to the
cells, but while the activation of T cells by the recipient’s increasing number of NK cells (Figure 1D). Altogether,
antigen-presenting cells often causes graft-versus-host dis- these experiments suggest that NK cells can negatively in-
ease (GvHD), a potentially lethal complication of the pro- fluence HSC frequency and function in culture.
cedure, NK cells are capable of eliminating tumor cells
without being involved in GvHD (Raziuddin et al., 2002; NK cells affect murine HSC repopulation capacity
Ruggeri et al., 2002). Thus, differently from T cells, NK cells in vivo
have been traditionally recognized as transplant helpers. Next, we investigated whether NK cells alter HSC proper-
However, the effects of donor NK cells based on their secre- ties in vivo. Sorted CD45.1/2 HSCs were cultured alone or
tory potential on HSC function during transplantation so co-cultured with CD45.2 NK cells (ratio of 1 3 105 NK cells
far remain unknown. to 1,500 HSCs) overnight, and injected into lethally irradi-
In the present study, we showed that NK cells impair HSC ated CD45.2 mice (Figure 2A). Importantly, we enumerated
function in culture and in vivo. Our results demonstrated HSCs post overnight culture and prior to transplantation,
that NK cells from the graft negatively influence HSC and observed a reduced number of remaining viable
engraftment and hematopoietic recovery upon BM trans- HSCs in cell suspensions containing NK cells compared

2000 Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021

 A B
p<0.001
CD45.2+ HSC 1.2

# HSC (Fold change)

Ly5.2 1
SCF + Flt3L + IL-3 + 0.8
BM IL-6 + TPO + IL-2
0.6
FACS
Day 4 HSCs 0.4

Ly5.1 OP9 stromal 0.2

cells
0
spleen HSC 1 1 1 1 1
CD45.1+ NK NK 0 6.25 12.5 25 50

C
BM HSC

SCF + Flt3L + IL-3 +

IL-6 + TPO + IL-2
O/N
incubation Day 10
Counting
colonies
Liquid culture
M3434 + IL-2

Spleen NK cells

D
p<0.0001
p=0.0038
(normalized to HSC)

1.2

0.8
# CFU

0.4

0.0
HSC HSC: HSC:
NK NK
1:10 1:50

Figure 1. NK cells reduce HSC maintenance and function in culture

(A) Graphical representation of the experimental design. Co-cultures were established in stem cell medium containing stem cell factor
(SCF), Flt3-ligand, IL-3, IL-6, thrombopoietin (TPO), and IL-2 over an OP9 stromal cell layer using sorted BM CD45.2+ HSCs (lin c-Kit+ Sca-
1 CD48 CD150+) and SP CD45.1+ NK cells (Ter119 CD19 CD4 CD8 CD3 NK1.1+). Days of culture are indicated. HSCs were enumerated
by FACS analysis.
(B) Quantification of HSCs recovered from 4-day cultures by FACS analysis. Y axis indicates the absolute number of HSCs. X axis indicates
presence (+) or absence () of NK cells in culture (ratios are indicated). Values and error bars indicate medians ±SEM. n = 3 biological
samples in each condition. Jonckheere-Terpstra trend test was used to assess statistical significance (p value is indicated).

(legend continued on next page)

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 2001

with suspensions without NK cells (Figure 2B). Nine weeks as well as long-term transplantation outcome without
after transplantation, peripheral blood analysis demon- altering the lineage hematopoietic recovery.
strated a reduction of CD45.1/2 cells when HSCs were
exposed to NK cells prior to transplantation (Figure 2C). NK cells negatively affect human CD34+ cells in vitro
However, NK-exposed HSCs showed no skewing to and in vivo
myeloid, B, or T cell differentiation (Figures 2D and S2A). Next, we assessed whether the effects that NK cells have on
Next, we assessed the long-term effects of exposing HSCs murine HSCs would translate to human primary samples.
to NK cells overnight, and observed a significantly reduced Human HSCs were isolated from BM (CD34+), co-cultured
percentage of CD45.1/2 cells in blood, BM, and spleen (SP) overnight in the presence of human IL-2, with or without
of recipient mice 16 weeks after transplantation (Figure 2E). human NK cells (CD19 CD8 CD4 CD3 CD56+), and
Importantly, homing assays demonstrated that addition of placed in semi-solid media supplemented with human
NK cells to the donor cell preparations did not alter HSC IL-2 (same strategy as in Figure 1C). The number of colonies
homing (Figure S2B). Together, these results demonstrated was enumerated at day 10 of culture, in which we observed
that NK cells negatively affect the numbers of functional colony-forming unit (CFU) reduction in the presence of NK
HSCs, and, therefore, reduce their hematopoietic reconsti- cells, suggesting that NK cells can harm human HSCs’ abil-
tution ability compared with non-exposed HSCs. ity to form colonies (Figure 4A). Since NK cell depletion
during murine BM transplantation assays improved he-
NK cells present in murine BM grafts compromise stem matopoietic engraftment and reconstitution, we next per-
cell function formed experiments to determine how depletion of NK
Since our previous results demonstrated that co-culturing cells from human grafts could affect HSCT. NK cell-
NK cells and HSCs lead to HSC impaired repopulation ca- depleted (CD56) or non-depleted (IgG) human BM
pacity, we hypothesized that NK cells and HSC co-habita- samples were transplanted into sublethally irradiated
tion in the BM donor samples could cause intrinsic changes immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG)
to HSC function. To test this hypothesis, we first assessed mice at different doses (Figure 4B). CD3 antibodies were
whether removal of NK cells from total BM would improve included in the depletion cocktail to remove human
short-term reconstitution. Whole BM from CD45.1 mice T cells from the graft, to prevent GvHD. The efficiency of
was submitted to immunomagnetic NK1.1 (NK cells) or immunomagnetic depletion of NK cells was demonstrated
immunoglobulin (Ig) G (control) depletion and injected by flow cytometry analysis prior to BM transplantation
into lethally irradiated CD45.2 mice (Figure 3A). NK cell (Figure S4). The engraftment and contribution of human
depletion was verified by flow cytometry analysis prior to BM cells to the hematopoietic system of recipient mice
transplantation (Figure S3A). Donor chimerism after was determined by the percentage of human CD45+ cells
8 weeks of transplant showed that NK cell depletion from in blood, BM, and SP 14 weeks after transplantation. We
the BM improved the short-term engraftment ability of observed that ablation of NK cells from human BM grafts
HSC (Figure 3B). Next, we performed limiting dilution had a positive effect in the transplants, leading to better
transplantation assays following the same NK cell depletion reconstitution and engraftment of human cells in recipient
strategy (Figure 3A), and assessed whether the long-term mice (Figure 4C). These positive effects after NK cell
HSC properties would be also affected by NK cells. Limiting removal were visible in different cell doses and in two inde-
dilution transplantation assays allowed us to measure the pendent experiments using different healthy BM grafts;
frequency of repopulating units, which reflects the number however, no statistical significance was reached and we
of functional HSCs. Positive engraftment was defined as could only observe a tendency in our human transplanta-
>0.3% and at least two-lineage reconstitution. We observed tion assays. Altogether, in vitro and in vivo experiments sug-
a 5-fold increased number of functional HSCs when NK gest that NK cells can impair HSC function, which can be
cells were removed prior to transplantation (p < 0.000804) restored by NK cell depletion.
(Figure 3C). Further, NK cell depletion from the BM graft
did not affect the differentiation pattern of lymphoid or Cebpg KO NK cells exhibit milder effects than wild-
myeloid lineages (Figures 3D and S3B). Altogether, our re- type NK cells on HSC function
sults demonstrated that NK cell removal from donor BM Next, since the transcription factor C/EBPg was related to
in murine transplantation assays improves the short-term NK cell function and Cebpg-deficient NK cells were severely

(C) Experimental design of in vitro colony culture assays.

(D) Quantification of colonies at day 10 of culture. Y axis indicates the number of CFU relative to the HSC counts. X axis indicates cells present in
the semi-solid cultures. Two different HSC/NK cell ratios were used as indicated. Data indicate mean ± SD of three independent biological
triplicates. Each dot represents one culture well. Two-tailed Student’s t test was used to assess statistical significance (p values are indicated).

2002 Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021

 A B
24-hour culture with IL-2 p=0.002
1000

# alive HSCs
800
1500 HSC 1500 HSC CD45.1/2 600
CD45.1/2 +1x105 NK CD45.2
400
Lethal irradiation 200
0
CD45.2 CD45.2 HSC + +
NK - +

C PB D
HSC HSC + NK
p=0.0032
50

Gr1/CD11b/B220

Gr1/CD11b/B220
% donor cells

CD45.2
40 19 62 9 69
CD45.2

30
20 17 20
10 31 10
0 CD45.1 CD3/B220
CD45.1 CD3/B220
HSC + +
NK - +

E PB BM SP
p=0.0091 p=0.03 p=0.0075
50 60 50
% donor cells

% donor cells

% donor cells

40 40
30 40 30
20 20 20
10 10
0 0 0
HSC + + HSC + + HSC + +
NK - + NK - + NK - +

Figure 2. NK cell exposure compromises HSC engraftment in vivo

(A) Experimental strategy for NK and HSCT assays. HSCs from C57BL/6 CD45.1/2 mice were purified by sorting, cultured in the presence or
absence of NK cells (Ter119 CD19 CD4 CD8 CD3 NK1.1+) obtained from C57BL/6 CD45.2 mice, and injected into lethally irradiated
CD45.2 recipient animals in a ratio of 105 NK cells to 103 HSCs per mouse.
(B) Number of HSCs assessed by FACS. Y axis indicates number of alive Hoechst 33,258 HSCs after overnight incubation. X axis indicates
culture conditions.
(C) Flow cytometry analysis of recipient mice peripheral blood 9 weeks after transplantation. Y axis indicates the percentage of donor-
derived CD45.1/2+ cells. X axis indicates different culture conditions.
(D) Representative flow cytometry plots of recipient mice peripheral blood 9 weeks after transplantation. Plots show CD45.1 versus CD45.2
expression in HSC and HSC + NK transplanted mice. Black box indicates the percentage of CD45.1/2+ donor-derived cells and the gate used
to analyze tri-lineage contribution: red box indicates the percentage of myeloid cells (Gr1/CD11b+), blue box the percentage of B cells
(B220+), and gray box the percentage of T cells (CD3+). See also Figure S2.
(E) FACS analysis of peripheral blood (PB), BM, and SP isolated from recipient mice 16 weeks after transplantation. Y axes indicate the
percentage of donor CD45.1/2+ cells and X axes indicate distinct culturing conditions. Five animals were included in each group. All data
represent mean ± SD from one representative experiment out of three. Two-tailed Student’s t test was used to assess statistical signifi-
cance (p values are indicated).

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 2003

 A B
BM
p=0.0025
80

% CD45.1+ cells
CD45.1 60

40
IgG control NK1.1
20
depletion depletion
0
IgG NK
control depletion
CD45.2
Lethal irradiation
C
IgG
% negative engraftment

100 NK depletion Injected cells (x 103) IgG NK depleted

37 20 1/5 2/5
50 3/11 5/11
10 250 4/6 4/5
1250 4/6 6/6
6250 6/6 6/6
1 Frequency 1 in 458 1 in 95
0 500 1000 1500 Confidence interval 95% 217-965 47-189
Chisq = 11.2; DF = 1; p = 0.000804
Number of cells transplanted

BM SP PB
Gr1 / CD11b / B220
IgG control

T B

M
CD45.2
NK depletion

CD45.1 CD3 / B220

Figure 3. NK cell depletion from murine BM improves stem cell function

(A) Overview of the experimental strategy for the BM transplantation assay after NK cell depletion (NK1.1) or irrelevant IgG depletion
(control). Upon depletion, cells were injected into lethally irradiated CD45.2 animals.
(B) FACS analysis of peripheral blood of recipient mice. Y axis indicates the percentage of donor CD45.1+ cells 9 weeks after trans-
plantation. X axis indicates IgG control or NK cell depletion. In each group 6.25 3 106 cells were transplanted into recipient mice. Six
animals were included in each group. All data represent mean ± SD from one representative experiment out of two. Two-tailed Student’s t
test was used to assess statistical significance (p value is indicated). See also Figure S3.

(legend continued on next page)

2004 Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021

impaired in their cytotoxicity against target cells (Kaisho T cells was similar in all three groups (Figure S5D). Together,
et al., 1999), we investigated the effect of Cebpg-deficient our in vitro and in vivo experiments demonstrated that
NK cells on HSC function. First, we analyzed Cebpg expres- Cebpg KO mice produce normal numbers of NK cells, which
sion in distinct hematopoietic populations, and observed are functionally impaired, and do have milder effects than
that Cebpg mRNA expression is higher in the lymphoid WT NK cells during HSCT.
than myeloid lineage and BM HSPCs (Figure 5A). The high-
est Cebpg mRNA expression was detected in NK cells. Next, The regulatory effects of NK cells on stem cells are
we took advantage of a Cebpg conditional KO mouse model cytokine mediated
generated in our laboratory (Kardosova et al., 2018). Cebpg Since Cebpg-deficient NK cells were not able to affect the he-
conditional KO mice were crossed to Vav-iCre transgenic matopoietic reconstitution of recipient mice, we investi-
mice in order to induce Cebpg excision in the hematopoiet- gated potential mediators of HSC regulation by NK cells
ic compartment. We generated Cebpgflox/flox Vav-iCre by comparing the gene expression profile of WT and Cebpg
(wild-type [WT]) and Cebpgflox/flox Vav-iCre+ (Cebpg KO) KO NK cells. NK cells were sorted from WT and Cebpg KO
mice. Absence of Cebpg was confirmed by mRNA quantifi- mice, cultured overnight with IL-2 or vehicle control, and
cation in BM and SP (Figure 5B). Frequency of Cebpg KO gene expression profiling was performed (E-MTAB-5604).
NK cells was not altered, and expression of NK cell-acti- Using the LIMMA package to assess differential gene
vating receptors was similar in KO and WT mice (Figures expression, we identified 1,074 genes deregulated in WT
S5A and S5B). However, purified Cebpg KO NK cells stimu- versus Cebpg KO NK cells (p < 0.05), and 1,316 genes de-
lated with IL-2 had lower cytotoxicity against murine YAC- regulated in IL-2-treated WT versus IL-2-treated Cebpg KO
1 lymphoma cells (Figure S5C), indicating that Cebpg-defi- NK cells (p < 0.05). Using prediction analysis of microar-
cient NK cells exhibit reduced functionality. Next, we rays, we identified and selected 84 probe sets, correspond-
assessed whether Cebpg KO NK cells would be able to affect ing to 75 genes, significantly up- or downregulated in WT
HSC colony-forming abilities. Interestingly, we observed and Cebpg KO NK cells in the presence or absence of IL-2
that the deleterious effect of NK cells was abolished when (Table S1). Figure 6A shows a heatmap and hierarchical
Cebpg was absent in NK cells, demonstrating that non- clustering according to expression of the 84 probe sets.
functional NK cells do not harm HSCs in vitro (Figure 5C). These results identified an NK signature characterized by
Further, we investigated whether Cebpg KO NK cells would a set of genes targeted upon NK activation. Pathway anal-
impair HSC function in vivo. Using similar HSC and NK cell ysis of differentially expressed mRNA demonstrated cell
co-culture conditions as previously described in Figure 2A, pathways associated to deregulated genes in WT versus
HSCs were isolated from WT CD45.1/2 mice and NK cells Cebpg KO NK cells (Table S2). Since we observed more pro-
were isolated from either WT or Cebpg KO mice (CD45.2), nounced effects on HSC function when NK cells were
co-cultured overnight, and were injected into lethally irra- exposed to IL-2, we further performed pathway analysis
diated CD45.2 recipients. Prior to transplantation, we in IL-2 treated samples. Our results demonstrated that the
enumerated HSCs in the co-cultures and observed that IFNg signaling pathway was the top pathway among de-
Cebpg KO NK cells do not have the ability to reduce the regulated genes, suggesting that it might contribute to
number of viable HSCs, in contrast to WT NK cells (Fig- NK cell effects on HSC (Table 1). To gain further insights
ure S5D). Flow cytometric analysis of recipient mice into the mechanisms mediating the NK cell effects, we
showed that the effect of Cebpg-deficient NK cells was analyzed differentially expressed genes between WT and
much milder than the effect of WT NK cells (Figure 5D). Cebpg-deficient NK cells treated with IL-2. Figure 6B shows
This partial rescue was present 9 and 16 weeks after trans- a heatmap and hierarchical clustering according to expres-
plantation, and the contribution to myeloid, B cells, and sion of 29 genes present in the NK signature. Within the

(C) Panels indicate results from limiting dilution competitive repopulation unit assays. (Left) Logarithmic plot showing the percentage of
negative recipients transplanted with different cell doses of murine BM depleted with NK1.1 Ab (black dots) or control IgG Ab (white dots).
Only recipients at 16 weeks with engraftment of CD45.1 cells R0.1% and contribution to all lineages (T cells, B cells, and granulocytes)
higher than 1% were considered responders. (Right) Table showing the number of responders and the total number of recipients trans-
planted per cell dose. Frequencies of HSCs (1:95 in NK1.1-depleted BM transplants versus 1:458 in IgG-depleted control, p = 0.000804)
were calculated according to Poisson statistics using ELDA software based on data from two independent experiments (Chisq, chi-square
test).
(D) Representative flow cytometry dot plots showing the percentages of CD45.1+ donor cells (blue boxes, Y axes) and CD45.2+ host cells (X
axes). Plots show BM, SP, and PB of mice injected with IgG-depleted control (upper panels) and NK-depleted (lower panels) BM cells
16 weeks after transplant. The panels on the right refer to gated CD45.1+ PB and indicate T (gray box), B (blue box), and myeloid (red box)
cells, as determined by the use of antibodies against CD3, B220, and Gr1/CD11b, respectively.

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 2005

 A p=0.0004 B
Negative
(normalized to HSC) 1.2
control
Human
0.8 (IgG control)
Bone
# CFU

NSG
Marrow
0.4 NK cell
depletion
0.0 (CD56 depletion)
CD34+ CD34+
C
NK
Donor 1

0.06 Dose 1 0.04 Dose 2 0.4 Dose 3

% of hCD45+ cells

0.03 0.3
0.04
(Blood)

0.02 0.2
0.02
0.01 0.1
0 0 0
IgG CD56 IgG CD56 IgG CD56
control depletion control depletion control depletion

Donor 2

Dose 1 Dose 2
cells

4 3
3
hCD45+

2
(BM)

2
1
1
% of

0 0
IgG CD56 IgG CD56
control depletion control depletion

Dose 1 Dose 2
% of hCD45+ cells

0.4 0.4
0.3 0.3
(SP)

0.2 0.2
0.1 0.1
0 0
IgG CD56 IgG CD56
control depletion control depletion

Figure 4. Engraftment of human BM stem cells is optimized by NK cell removal

(A) Human CFU assays of CD34+ cells with or without NK cells (ratio 1:10), after overnight culture in the presence of human IL-2. Y axis
indicates the mean CFU number ±SD from two distinct human BM samples relative to CD34+ cells cultured alone. X axis indicates the
distinct culture conditions. Each black dot indicates values for one culture well. Two-tailed Student’s t test was used to assess statistical
significance (p value is indicated).
(B) Illustration of the experimental strategy. Human BM samples depleted with antibodies against CD3 and IgG (negative control) or
against CD3 and CD56 (NK cell depletion) were transplanted into sublethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice at
different doses.

(legend continued on next page)

2006 Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021

upregulated genes in the Cebpg KO NK cells, we identified DISCUSSION
lactotransferrin (Ltf), lysozyme 1 (Lyz1), granzyme M
(Gzmm), and interferon-induced transmembrane proteins Cellular crosstalk between distinct cell types occurs in our
1, 3, and 6 (Ifitm1, Ifitm3, and Ifitm6). Within the downre- organism, including hematopoietic cells. Of particular in-
gulated genes in the Cebpg KO NK cells, we identified inter- terest is the relationship that HSCs establish with their
feron regulatory factor 2-binding protein (Irf2pb1), vascular neighbor cells (Guezguez et al., 2013; Hoggatt et al.,
endothelial growth factor A (Vegfa), and IL-15 receptor 2016; Mirantes et al., 2014; Schuettpelz and Link, 2013;
alpha chain (Il15ra). Schurch et al., 2014). During the last years it became
In parallel, supernatants from the overnight cultures evident that this crosstalk not only regulates normal hema-
were analyzed by a flow cytometry-based cytokine bead topoiesis but also affects the outcome of certain clinical in-
array, which allows identification and quantification of a terventions, such as BM transplantation (Crippa and Ber-
panel of cytokines. We observed that, upon NK cell activa- nardo, 2018; Russell et al., 2015; Touzot et al., 2015;
tion, IFNg production was diminished in Cebpg-deficient Triplett et al., 2015). In the present study, we showed that
NK cells in comparison with WT NK cells (Figure 6C). Alto- NK cells, an important component of the innate immune
gether, our findings identified a list of genes and pathways system, negatively affect HSC function during transplanta-
potentially mediating the negative NK cell effects on HSC tion, and that this detrimental effect can be ameliorated by
function, and suggested that IFNg production by NK cells either (1) removing NK cells from the donor graft, or (2) by
may negatively affect HSCs during transplantation. blocking IFNg signaling during HSCT. Previously, the role
of NK cells in HSCT has largely been addressed, but with
IFNg-neutralizing antibody restores HSC function in a different focus than ours. The majority of reports focused
the presence of WT NK cells in vitro and in vivo on the ability of donor NK cells to recognize and kill resid-
Based on our observations, we first assessed whether IFNg- ual tumor cells; mediate innate immune responses to pre-
neutralizing antibody would rescue the deleterious effect vent post-transplant infections; facilitate adaptive immune
of NK cells on HSCs in culture. We observed that the num- responses mediated by B and T cells; lyse host dendritic
ber of colonies was restored when IFNg-neutralizing anti- cells, thus reducing the risk of GvHD; and contribute to
body was added to the WT NK-containing cultures, while epithelial regeneration (Leung, 2011; Montaldo et al.,
the presence of IFNg-neutralizing antibody did not alter col- 2013; Palmer et al., 2013; Parham and McQueen, 2003;
ony numbers with or without Cebpg KO NK cells (Figure 6D). Passweg et al., 2004). In contrast to these studies support-
Since humanized anti-IFNg monoclonal antibodies are ing the influence of NK cells from BM grafts on host cells,
available and ready for clinical use, we next investigated our data show that donor NK cells can also directly target
whether inhibition of the IFNg signaling pathway could donor HSCs and affect their reconstitution potential.
improve short-term engraftment, a critical phase during hu- C/EBPg is a transcription factor known to participate in
man HSCT. G-CSF mobilized peripheral blood was trans- the maturation and function of NK cells (Di Santo, 2006;
planted into NSG mice, and, 4 h after transplantation, treat- Kaisho et al., 1999). Recently, we generated a conditional
ment with either IFNg-blocking antibodies or IgG control KO mouse model with specific excision of Cebpg in hemato-
was initiated and continued as indicated in Figure 6E. We poietic cells (Kardosova et al., 2018). Here, we confirmed the
observed that mice treated with IFNg-blocking antibodies previously reported functional defects in Cebpg KO NK cells
exhibited improved percentage and absolute numbers of (Kaisho et al., 1999), and used this model to investigate the
human CD45+ cells in BM compared with mice treated potential mechanisms of NK-HSC regulation. Our data
with IgG control antibody (Figure 6F). Further, we observed demonstrate that Cebpg-deficient NK cells, which do not
that animals that received IFNg-blocking treatment showed harm HSC fitness, produce reduced levels of IFNg in com-
increased relative and absolute numbers of human CD34+ parison with WT NK cells. Accordingly, the use of IFNg-
cells in the BM (Figure 6G). Together, our data suggest neutralizing antibodies in culture as well as during HSCT
that blocking IFNg improves HSC function in vitro and improved HSC function. Thus, our results suggest that the
engraftment during the early phase of HSCT. INFg secreted by NK cells contributes to the detrimental

(C) The percentage of human CD45+ donor cells in murine blood, BM, and SP determined 14 weeks after transplantation. Results for two
independent experiments using two healthy BM donors (donor 1 and donor 2) are shown. Y axes indicate the percentage of human CD45+
cells in the indicated tissues. Black boxes indicate animals transplanted with IgG-depleted control BM and red boxes indicate animals
transplanted with NK-depleted (CD56 depletion) BM. Transplanted cell doses are indicated as dose 1 (0.5 3 106), dose 2 (1 3 106), and
dose 3 (2.5 3 106). Data represent mean ± SD from two independent experiments. Two-tailed Student’s t test was used to assess statistical
significance, p values were not statistically significant.
See also Figure S4.

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 2007

 A

B C

Figure 5. Defective Cebpg KO NK cells exhibit milder effects on HSC function than WT NK cells
(A) Relative Cebpg expression levels shown as the percentage of Gapdh (% Gapdh) in distinct sorted hematopoietic cell populations. LT
(long-term HSC), MPP (multi-potent progenitor), CMP (common myeloid progenitor), GMP (granulocyte-macrophage progenitor), MEP
(megakaryocyte-erythroid progenitor), CLP (common lymphoid progenitor), Gran (granulocyte), Gr1lo Mon (Gr1lo monocyte), Gr1hi Mon
(Gr1hi monocyte), B cells, T CD4+ cells, T CD8+cells, and NK cells.
(B) Cebpg mRNA levels in BM and SP isolated from WT and Cebpg KO mice. Y axis indicates relative Cebpg expression levels relative to Gapdh
(% Gapdh).
(legend continued on next page)

2008 Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021

effects on HSC. Accordingly, previous studies suggested ence of NK cells in the BM microenvironment can modu-
negative effects of IFNg on HSC function. For instance, late HSC activity. However, further studies will need to
enhanced proliferation of HSCs and reduction in long- address whether the co-existence of these cell types in
term repopulating capacity was associated with IFNg stimu- the BM niche during steady-state hematopoiesis may
lation of murine cells in experimental models of bacterial modulate HSC fitness.
infection (Baldridge et al., 2010; MacNamara et al., 2011). It is important to consider that NK cell activity during
In agreement, Yang et al. (2005) showed that the ability of HSCT may differ depending on the origin of the NK cells
CD34+CD38 human cord blood cells to support hemato- (i.e., recipient NK versus donor NK cells). Recipient NK cell
poiesis was inhibited by IFNg treatment in a xenotransplant subsets bearing MHC class I receptors are said to be licensed
model. However, IFNg is not exclusively produced by NK or educated, and rapidly respond to stimuli (Alvarez et al.,
cells, and other immune cells, such as T cells, can also pro- 2016). While licensed recipient NK subsets have been impli-
duce INFg and, consequently, contribute to the deleterious cated in inhibition of allogenic BM cells and consequent
effects on HSCs. In fact, T cells produce IFNg and TNFa, rejection, unlicensed recipient NK cells seem to be growth
and it was recently suggested that neutralizing T cell-medi- promoting, thus facilitating BM engraftment (Sun et al.,
ated TNFa signaling was able to enhance engraftment and 2012). In the clinical setting, it is undeniable that NK cells
hematopoietic reconstitution upon HSCT (Wang et al., play a relevant role in promoting the clearance of malignant
2017). Together, these studies seed the path to potentially cells and avoiding HSCT complications, particularly in HLA-
novel strategies directed to enhance and improve HSCT, in haploidentical transplants (Di Santo, 2006). Here, we
particular during the critical early stages upon transplanta- describe a novel non-HLA-dependent ’graft NK’ - ’graft
tion. Further studies may address whether anti-inflamma- HSC’ regulation mechanism that may influence transplant
tory regimes shortly after transplantation, and for a limited outcome. The presence of NK cells in the graft, which are
period of time, may improve HSCT. also transplanted and activated during the graft product
In our assays, since HSCs and mature NK cells were both infusion, may harm HSCs contained in the graft regardless
isolated from the same mouse strain that differed only in of the ’missing self’ recognition mechanism, and therefore
CD45.1 or CD45.2 expression (congenic mice), no differ- impair engraftment. It is certainly beneficial to have NK
ential expression of MHC class I ligands was expected. donor cell-rich preparations for malignant diseases such as
Therefore, we assumed that the observed effects were asso- acute leukemias because of the desired graft-versus-leukemia
ciated with NK secretory function rather than being (GvL) effect. However, we would like to propose that NK cell
dependent on the classic NK missing self-recognition depletion may actually favor HSC donor engraftment in the
function. Accordingly, gene expression analysis of Cebpg- setting of allogeneic transplantation for non-neoplastic dis-
deficient NK cells, compared with controls, revealed eases in selected patients. Such cases would include for
deregulation of genes from several cytokine pathways. It instance, but are not limited to, patients with idiopathic se-
was previously reported that, when BM cells were placed vere aplastic anemia or sickle cell anemia. For those situa-
under ideal culture conditions, the addition of activated tions where GvL effect is not needed, having highly func-
NK cells to the culture inhibited colony formation (Mur- tional donor HSCs may accelerate blood recovery and
phy et al., 1992). Our overnight co-cultures followed by increase the chances of sustained donor engraftment. NK
frequency and functional assessment indicated reduced depletion could gain great value when clinicians deal with
HSC numbers and activity in the presence of NK cells, limited donor cell numbers. Low number of HSCs in BM
which could be at least partially explained by a reduction preparations can be a problem when insufficient HLA-
in viability. However, these effects were lost when Cebpg matched cord blood sources are available for adult patients,
KO NK cells were employed. Together, based on our obser- such as in geographical regions where mixed ethnicity im-
vations on the crosstalk between HSCs and NK cells in pairs HLA-matched donor availability. Also, peripheral
culture and during HSCT, we hypothesize that the pres- blood mobilized cells (PBMCs) used for autologous

(C) Colony culture assays of murine cells. Y axis indicates the number of CFU relative to HSC condition. X axis indicates culture conditions:
HSC alone or in the presence of WT or Cebpg KO NK cells. Cell ratio is indicated. Each dot represents one culture dish. Data represent mean ±
SD from three independent experiments.
(D) Engraftment of CD45.1/2 cells in blood of recipient CD45.1 mice evaluated 9 (left) and 16 weeks (right) after transplantation. X axis
indicates transplant conditions where purified HSCs alone or with WT or Cebpg KO NK cells were co-cultured overnight in the presence IL-2
prior to transplantation. Y axis demonstrates the percentage of CD45.1/2 cells. Each dot indicates values for one animal. All data represent
mean ± SD from two independent experiments. For (C) and (D), two-tailed Student’s t test was used to assess statistical significance (p
values are indicated; ns, not significant).
See also Figure S5.

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 2009

 A D

Figure 6. Cebpg deletion results in reduced IFNg production in NK cells and blocking IFNg signaling improves HSC fitness
(A) Heatmap and hierarchical clustering based on gene expression of 84 probe sets, which compose the NK signature. NK cells were isolated
from WT and Cebpg KO murine SPs in non-stimulation conditions or upon stimulation with the NK cell-activating cytokine IL-2 (n = 4 for
each condition). Data were normalized to z scores for each gene. Red/blue color indicates increase/decrease in gene expression relative to
the universal mean for each gene. See also Table S1.
(legend continued on next page)

2010 Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021

 by our group and backcrossed into the C57BL/6 background
Table 1. Cebpg ablation results in differential pathway
(Kardosova et al., 2018). Cebpgflox/flox (Cebpgf/f) mice were bred
enrichment analysis in WT and Cebpg KO NK cells stimulated
to Vav-iCre transgenic mice (also in C57BL/6 background) to
with IL-2
generate Cebpgf/f Vav-iCre mice (control, WT) or Cebpgf/f
Combined Vav-iCre+ (KO with Cebpg specifically excised in the hematopoietic
Index Name p value Z score score
system), and used for experimental comparisons in this study.
1 interferon-gamma signaling 0.01414 1.57 1.56 Transplantations of human samples were performed on NSG
pathway mice (Jackson Laboratory, stock no. 005557), which were main-
tained in specified pathogen-free conditions. All mice were housed
2 insulin/IGF pathway- 0.01625 1.47 1.46
mitogen in a sterile barrier facility.
activated protein kinase
kinase/MAP kinase cascade Isolation of murine NK and HSC cells
NK cells were obtained from SP from WT C56BL/6, Cebpgf/f Vav-
3 apoptosis signaling pathway 0.01821 1.40 1.39
iCre, and Cebpgf/f Vav-iCre+ (CD45.2+) mice. HSCs were obtained
4 oxidative stress response 0.02026 0.78 0.77 from WT C57BL/6 CD45.2 or C57BL/6 CD45.1/CD45.2 mice. SP
cell suspensions were obtained and submitted to red blood cell
5 T cell activation 0.02227 1.20 1.20
(RBC) lysis, followed by immunomagnetic lineage depletion using
Results of Panther pathway analysis of IL-2-treated WT versus Cepbg KO NK Ter119, CD4, CD8, and CD19 biotinylated antibodies (BioLegend)
cells with the threshold of p < 0.05. and Anti-Biotin Microbeads Ultrapure (Miltenyi Biotec), and then
sorted by flow cytometry as lineage negative, CD3 negative, and
NK1.1 positive (Lin, CD3, NK1.1+). To obtain RNA and cell super-
transplantation in cancer patients undergoing aggressive
natants, 2.5 3 105 NK cells were cultured in RPMI1640 supple-
therapy frequently have low numbers of HSCs.
mented with 10% FBS and 1000 U/mL of IL-2 for 24 h. To isolate
Future prospective studies are needed to investigate HSCs, BM samples were lysed by RBC lysis buffer and subsequently
whether NK frequency in HSC donor sources may be depleted using antibodies against Ter-119, CD19, B220, CD8, Gr1,
proved to be an additional non-HLA risk factor for BM and CD11b antigens by immunomagnetic separation, and then
transplantation outcomes together with recipient age, sorted as c-Kithi, Sca-1+, lineage-negative, CD48, CD150+ cells. Cul-
stage of disease, cytomegalovirus (CMV) serostatus, and co- ture conditions are detailed in the online supplemental information.
morbidities. If this is true, improving HSC function in bone
marrow grafts by reducing the effect of NK cells can be clin- Flow cytometry
ically important to make BM transplantation feasible for Single-cell suspensions from murine PB, BM, or SP were analyzed
individualized selected patients and to accelerate engraft- by flow cytometry using the following monoclonal antibodies
ment in adverse conditions for both allogeneic and autolo- conjugated with biotin (BIO), fluorescein isothiocyanate (FITC),
gous transplantation. phycoerythrin (PE), PE-Cy5, PE–Cy7, Pacific Blue (PB), allophyco-
cyanin (APC), or APC–Cy7 and obtained from BioLegend or
eBioscience: CD19 (MB19-1), B220 (RA3-6B2), CD4 (RM4-5),
EXPERIMENTAL PROCEDURES CD8 (53–6.7), Gr1 (RB6-8C5), CD11b (M1/70), TER119 (TER-
119), Sca-1 (D7), streptavidin, c-Kit (2B8), CD48 (HM48-1),
Mice CD150 (TC15-12F12.2), CD3 (17A2), NK1.1 (PK136), NKG2A
The cell subsets used for this study were obtained from WT C57BL/ (20d5), NKG2D (CX5), Ly-49H (3D10), CD45.1 (A20), and
6 CD45.1, C57BL/6 CD45.2, or C57BL/6 CD45.1/CD45.2 congenic CD45.2 (104). Stained cells were analyzed with an LSRII flow cy-
mouse strain. A Cebpg conditional KO mouse model was generated tometer and sorted using a FACSAria II or Influx (BD Biosciences).

(B) Heatmap and hierarchical clustering according to expression of 29 genes present in the NK signature. WT and Cepbg KO NK cells were
treated with IL-2 prior to gene expression profile analysis. Several genes deregulated between the two groups are indicated.
(C) IFNg levels in supernatants after culturing overnight WT or Cebpg KO NK cells. Cultures were established in the absence or presence of
IL-2. Y axes indicate cytokine levels (pg/mL) related to the condition without IL-2 for WT and KO NK cells (fold change). n = 4 mice per
group, two independent experiments.
(D) Colony culture assays of murine cells. Y axes indicate the number of CFU. X axes indicate culture conditions: NK cells correspond to WT
or Cebpg KO mice as indicated. Each dot represents one culture well, two independent experiments were performed.
(E) Illustration of the experimental scheme. Arrows and numbers indicate days when treatment was administered. Seventeen days after
transplantation, recipient NSG mice were sacrificed and analyzed.
(F and G) Relative percentage (left graphics) and absolute number of cells per femur (right graphics). Y axes indicate the percentage (%)
and numbers (#) of human CD45+ cells (F) and CD34+ cells (G) in BM. Black boxes indicate values for mice that received treatment with
mouse IgG control antibodies, and red boxes indicate values for mice that received IFNg-blocking antibody treatment. Each animal is
indicated by a symbol (n = 5–6 animals per group). For (C), (D), (F) and (G), data represent mean ± SD, two-tailed Student’s t test was used
to assess statistical significance (p values are indicated; ns, not significant).

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 2011

Viable cells were identified by Hoechst 33258 exclusion. Diva (BD Center grant MOE2014-T3-1-006 from the NRF and MOE,
Biosciences) and FlowJo (Tree Star) software were used for data Singapore, and NIH grants R35CA197697 and P01HL131477.
acquisition and analysis, respectively. CountBright Absolute The authors thank Prof. Vaclav Horejsi for providing us with the
Counting Beads (Molecular probes, Invitrogen) were used for IFNg and IgG1 antibodies.
HSC subpopulation quantification by flow cytometry.
Received: October 24, 2019
Study approval Revised: June 10, 2021
Animal studies were approved by the Institutional Animal Care Accepted: June 10, 2021
and Use Committee (IACUC) at the Beth Israel Deaconess Medical Published: July 8, 2021
Center (Boston, United States) and by the Animal Ethical Commit-
tee at the Institute of Molecular Genetics (Prague, Czech Republic). REFERENCES
Human BM samples were obtained after written informed consent
Alvarez, M., Sun, K., and Murphy, W.J. (2016). Mouse host unli-
was received from participants. Human studies were approved by
censed NK cells promote donor allogeneic bone marrow engraft-
the institutional review board at the First Faculty of Medicine,
ment. Blood 127, 1202–1205.
Charles University in Prague and General University Hospital
(Prague, Czech Republic). Baldridge, M.T., King, K.Y., Boles, N.C., Weksberg, D.C., and Goodell,
M.A. (2010). Quiescent haematopoietic stem cells are activated by
IFN-gamma in response to chronic infection. Nature 465, 793–797.
Statistical analysis
Statistical significance for indicated datasets was determined using Buza-Vidas, N., Antonchuk, J., Qian, H., Mansson, R., Luc, S.,
two-sided, unpaired Student’s t test, and p values < 0.05 were Zandi, S., Anderson, K., Takaki, S., Nygren, J.M., Jensen, C.T.,
considered statistically significant. Scatter dot plots depict mean et al. (2006). Cytokines regulate postnatal hematopoietic stem
with error bars representing standard deviation (SD). Jonckheere- cell expansion: opposing roles of thrombopoietin and LNK. Genes
Terpstra trend tests (for group comparisons) using the SPSS soft- Dev. 20, 2018–2023.
ware version 20 was employed when indicated. HSC frequencies Chen, C., Busson, M., Rocha, V., Appert, M.L., Lepage, V., Dulphy,
were calculated with L-Calc software (StemCell Technologies, Van- N., Haas, P., Socie, G., Toubert, A., Charron, D., et al. (2006). Acti-
couver, Canada) using Poisson statistics and the method of vating KIR genes are associated with CMV reactivation and survival
maximum likelihood to the proportion of negative recipients in after non-T-cell depleted HLA-identical sibling bone marrow trans-
a limiting dilution setting. plantation for malignant disorders. Bone Marrow Transplant. 38,
437–444.
Data and code availability Crippa, S., and Bernardo, M.E. (2018). Mesenchymal stromal cells:
The accession number for the microarray data reported in this pa- role in the BM niche and in the support of hematopoietic stem cell
per is ArrayExpress: E-MTAB-5604. transplantation. Hemasphere 2, e151.
Di Santo, J.P. (2006). Natural killer cell developmental pathways: a
SUPPLEMENTAL INFORMATION question of balance. Annu. Rev. Immunol. 24, 257–286.
Geerman, S., Brasser, G., Bhushal, S., Salerno, F., Kragten, N.A.,
Supplemental information can be found online at https://doi.org/
Hoogenboezem, M., de Haan, G., Wolkers, M.C., Pascutti, M.F.,
10.1016/j.stemcr.2021.06.008.
and Nolte, M.A. (2018). Memory CD8(+) T cells support the main-
tenance of hematopoietic stem cells in the bone marrow. Haema-
AUTHOR CONTRIBUTIONS
tologica 103, e230–e233.
Conceptualization, L.L.d.F-P., R.S.W., D.G.T., and M.A.-J.; method- Geerman, S., and Nolte, M.A. (2017). Impact of T cells on hemato-
ology, L.L.d.F-P., R.S.W, S.L., and M.A.-J.; formal analysis, L.L.d.F-P., poietic stem and progenitor cell function: good guys or bad guys?
H.S., and M.A.-J.; investigation, L.L.d.F-P., R.S.W, M.K.A., S.G., World J. Stem Cells 9, 37–44.
M.K., I.A.L., A.F.d.O.C., H.Z., A.M., A.T.J., M.Z., and M.A.-J.; data
Guezguez, B., Campbell, C.J., Boyd, A.L., Karanu, F., Casado, F.L.,
curation, H.S.; writing, L.L.d.F-P. and M.A.-J.; visualization,
Di Cresce, C., Collins, T.J., Shapovalova, Z., Xenocostas, A., and
L.L.d.F-P. and M.A.-J.; supervision, L.L.d.F-P., R.S.W., D.G.T., and
Bhatia, M. (2013). Regional localization within the bone marrow
M.A.-J.; funding acquisition, L.L.d.F-P., D.G.T., and M.A.-J.
influences the functional capacity of human HSCs. Cell Stem
Cell 13, 175–189.
DECLARATION OF INTERESTS
Hirata, Y., Furuhashi, K., Ishii, H., Li, H.W., Pinho, S., Ding, L., Rob-
The authors declare no competing interests. son, S.C., Frenette, P.S., and Fujisaki, J. (2018). CD150(high) bone
marrow Tregs maintain hematopoietic stem cell quiescence and
ACKNOWLEDGMENTS immune privilege via adenosine. Cell Stem Cell 22, 445–453.
This work was supported by a grant from São Paulo Research Foun- Hoggatt, J., Kfoury, Y., and Scadden, D.T. (2016). Hematopoietic stem
dation (FAPESP; grant 2015/21866-1) to L.L.d.F.-P. M.A.-J. was sup- cell niche in health and disease. Annu. Rev. Pathol. 11, 555–581.
ported by a GACR grant 18-08577S and institutional funding from Joncker, N.T., Shifrin, N., Delebecque, F., and Raulet, D.H. (2010).
the IMG CAS (RVO 68378050). D.G.T. was supported by a STaR Mature natural killer cells reset their responsiveness when exposed
Investigator Award, an RCE Core grant, and Tier 3 RNA Biology to an altered MHC environment. J. Exp. Med. 207, 2065–2072.

2012 Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021

Kaisho, T., Tsutsui, H., Tanaka, T., Tsujimura, T., Takeda, K., Kawai, Schuettpelz, L.G., and Link, D.C. (2013). Regulation of hematopoi-
T., Yoshida, N., Nakanishi, K., and Akira, S. (1999). Impairment of etic stem cell activity by inflammation. Front. Immunol. 4, 204.
natural killer cytotoxic activity and interferon gamma production Schurch, C.M., Riether, C., and Ochsenbein, A.F. (2014). Cytotoxic
in CCAAT/enhancer binding protein gamma-deficient mice. CD8+ T cells stimulate hematopoietic progenitors by promoting
J. Exp. Med. 190, 1573–1582. cytokine release from bone marrow mesenchymal stromal cells.
Kardosova, M., Zjablovskaja, P., Danek, P., Angelisova, P., de Figueir- Cell Stem Cell 14, 460–472.
edo-Pontes, L.L., Welner, R.S., Brdicka, T., Lee, S., Tenen, D.G., and Al- Storek, J., Gooley, T., Witherspoon, R.P., Sullivan, K.M., and Storb,
berich-Jorda, M. (2018). C/EBPgamma is dispensable for steady-state R. (1997). Infectious morbidity in long-term survivors of allogeneic
and emergency granulopoiesis. Haematologica 103, e331–e335. marrow transplantation is associated with low CD4 T cell counts.
Kim, S., Poursine-Laurent, J., Truscott, S.M., Lybarger, L., Song, Y.J., Am. J. Hematol. 54, 131–138.
Yang, L., French, A.R., Sunwoo, J.B., Lemieux, S., Hansen, T.H., Sun, K., Alvarez, M., Ames, E., Barao, I., Chen, M., Longo, D.L., Re-
et al. (2005). Licensing of natural killer cells by host major histo- delman, D., and Murphy, W.J. (2012). Mouse NK cell-mediated
compatibility complex class I molecules. Nature 436, 709–713. rejection of bone marrow allografts exhibits patterns consistent
Leung, W. (2011). Use of NK cell activity in cure by transplant. Br. J. with Ly49 subset licensing. Blood 119, 1590–1598.
Haematol. 155, 14–29. Tomblyn, M., Young, J.A., Haagenson, M.D., Klein, J.P., Trachten-
MacNamara, K.C., Jones, M., Martin, O., and Winslow, G.M. berg, E.A., Storek, J., Spellman, S.R., Cooley, S., Miller, J.S., and
(2011). Transient activation of hematopoietic stem and progenitor Weisdorf, D.J. (2010). Decreased infections in recipients of unre-
cells by IFNgamma during acute bacterial infection. PLoS one 6, lated donor hematopoietic cell transplantation from donors with
e28669. an activating KIR genotype. Biol. Blood Marrow Transplant. 16,
Mehta, R.S., Shpall, E.J., and Rezvani, K. (2015). Cord blood as a 1155–1161.
source of natural killer cells. Front. Med. 2, 93. Touzot, F., Neven, B., Dal-Cortivo, L., Gabrion, A., Moshous, D.,
Mirantes, C., Passegue, E., and Pietras, E.M. (2014). Pro-inflamma- Cros, G., Chomton, M., Luby, J.M., Terniaux, B., Magalon, J.,
tory cytokines: emerging players regulating HSC function in et al. (2015). CD45RA depletion in HLA-mismatched allogeneic
normal and diseased hematopoiesis. Exp. Cel. Res. 329, 248–254. hematopoietic stem cell transplantation for primary combined im-
Montaldo, E., Vacca, P., Moretta, L., and Mingari, M.C. (2013). Un- munodeficiency: a preliminary study. J. Allergy Clin. Immunol.
derstanding human NK cell differentiation: clues for improving 135, 1303–1309 e1301.
the haploidentical hematopoietic stem cell transplantation. Im- Triplett, B.M., Shook, D.R., Eldridge, P., Li, Y., Kang, G., Dallas, M.,
munol. Lett. 155, 2–5. Hartford, C., Srinivasan, A., Chan, W.K., Suwannasaen, D., et al.
Murphy, W.J., Keller, J.R., Harrison, C.L., Young, H.A., and Longo, (2015). Rapid memory T-cell reconstitution recapitulating CD45RA-
D.L. (1992). Interleukin-2-activated natural killer cells can support depleted haploidentical transplant graft content in patients with he-
hematopoiesis in vitro and promote marrow engraftment in vivo. matologic malignancies. Bone Marrow Transplant. 50, 1012.
Blood 80, 670–677. Venstrom, J.M., Gooley, T.A., Spellman, S., Pring, J., Malkki, M.,
Palmer, J.M., Rajasekaran, K., Thakar, M.S., and Malarkannan, S. Dupont, B., Petersdorf, E., and Hsu, K.C. (2010). Donor activating
(2013). Clinical relevance of natural killer cells following hemato- KIR3DS1 is associated with decreased acute GVHD in unrelated
poietic stem cell transplantation. J. Cancer 4, 25–35. allogeneic hematopoietic stem cell transplantation. Blood 115,
Parham, P., and McQueen, K.L. (2003). Alloreactive killer cells: hin- 3162–3165.
drance and help for haematopoietic transplants. Nat. Rev. Immu- Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S.
nol. 3, 108–122. (2008). Functions of natural killer cells. Nat. Immunol. 9, 503–510.
Passweg, J.R., Tichelli, A., Meyer-Monard, S., Heim, D., Stern, M., Wagner, J.E., Thompson, J.S., Carter, S.L., and Kernan, N.A. (2005).
Kuhne, T., Favre, G., and Gratwohl, A. (2004). Purified donor NK- Effect of graft-versus-host disease prophylaxis on 3-year disease-
lymphocyte infusion to consolidate engraftment after haploident- free survival in recipients of unrelated donor bone marrow (T-cell
ical stem cell transplantation. Leukemia 18, 1835–1838. Depletion Trial): a multi-centre, randomised phase II-III trial. Lan-
Raziuddin, A., Longo, D.L., Bennett, M., Winkler-Pickett, R., cet 366, 733–741.
Ortaldo, J.R., and Murphy, W.J. (2002). Increased bone marrow allo- Wang, W., Fujii, H., Kim, H.J., Hermans, K., Usenko, T., Xie, S., Luo,
graft rejection by depletion of NK cells expressing inhibitory Ly49 Z.J., Ma, J., Celso, C.L., Dick, J.E., et al. (2017). Enhanced human he-
NK receptors for donor class I antigens. Blood 100, 3026–3033. matopoietic stem and progenitor cell engraftment by blocking
Ruggeri, L., Capanni, M., Urbani, E., Perruccio, K., Shlomchik, donor T cell-mediated TNFalpha signaling. Sci. Transl. Med. 9,
W.D., Tosti, A., Posati, S., Rogaia, D., Frassoni, F., Aversa, F., et al. eaag3214.
(2002). Effectiveness of donor natural killer cell alloreactivity in Yang, L., Dybedal, I., Bryder, D., Nilsson, L., Sitnicka, E., Sasaki, Y.,
mismatched hematopoietic transplants. Science 295, 2097–2100. and Jacobsen, S.E. (2005). IFN-gamma negatively modulates self-
Russell, A., Malik, S., Litzow, M., Gastineau, D., Roy, V., and renewal of repopulating human hemopoietic stem cells.
Zubair, A.C. (2015). Dual roles of autologous CD8+ T cells in J. Immunol. 174, 752–757.
hematopoietic progenitor cell mobilization and engraftment. Zhang, C.C., and Lodish, H.F. (2008). Cytokines regulating he-
Transfusion 55, 1758–1765, quiz 1757. matopoietic stem cell function. Curr. Opin. Hematol. 15, 307–311.

Stem Cell Reports j Vol. 16 j 1999–2013 j August 10, 2021 2013

Stem Cell Reports, Volume 16

Supplemental Information

Improved hematopoietic stem cell transplantation upon inhibition of

natural killer cell-derived interferon-gamma
Lorena Lobo de Figueiredo-Pontes, Miroslava K. Adamcova, Srdjan Grusanovic, Maria
Kuzmina, Izabela Aparecida Lopes, Amanda Fernandes de Oliveira Costa, Hong
Zhang, Hynek Strnad, Sanghoon Lee, Alena Moudra, Anna T. Jonasova, Michal
Zidka, Robert S. Welner, Daniel G. Tenen, and Meritxell Alberich-Jorda
Supplemental Table of Contents

Figure S1. NK cells reduce HSC maintenance and frequency in culture. Related to figure 1.
Figure S2. Donor cell contribution to production of T,- B-, myeloid cells and homing assays.
Related to figure 2.
Figure S3. NK cell depletion from murine BM grafts improves hematopoietic recovery of
recipient mice. Related to figure 3.
Figure S4. Human bone marrow fractionation. Related to figure 4.
Figure S5. Characterization and function of Cebpg KO NK cells in comparison to WT NK
cells. Related to figure 5.
Table S1. List of genes belonging to the NK cell signature. Related to Figure 6.
Table S2. Differential pathway enrichment analysis in WT and Cebpg KO NK cells. Related
to Figure 6.

Supplemental Experimental Procedures

Supplemental References

Supplemental Information Page 1

 HSC NK:HSC 6.25:1 NK:HSC 12.5:1

NK:HSC 25:1 NK:HSC 50:1 NK:HSC 100:1

Figure S1. NK cells reduce HSC maintenance and frequency in culture. Related to figure 1. Images of
HSC:NK 4-day co-cultures in the presence of IL-2. Pictures were taken by microscopy of the tissue culture
plates (100 X). HSC:NK cell ratio is indicated. Arrows indicate non-viable colonies. Notice the reduction in
colonies with increasing amounts of NK cells in culture.

Supplemental Information Page 2

A
100
% donor cells

T cells
50 Myeloid cells
B cells

0
1 2 3 4 5 6 7 8 9 10
HSC: alone + NK cells

B
ns ns
% donor KSL cells

1.5
1
% donor lin- cells

0.75 1
0.5
0.5
0.25
0 0
LKS+NK
LKS

LKS
LKS+NK

Figure S2. Donor cell contribution to production of T,- B-, myeloid cells and homing assays. Related
to figure 2. (A) Blood sample analysis nine weeks post-transplantation. The bars show the average
percentages of donor CD45.1/2 cells expressing myeloid (Gr-1 and CD11b), B- (B220), or T- (CD4/8) cell
lineage markers. 1-5 indicates mice transplanted with HSC, and 6-10 indicates mice transplanted with HSC
exposed to NK cells. (B) Lineage- c-Kit+ Sca-1+ (LKS) cells (CD45.1) were injected into sublethally
irradiated CD45.1/2 mice (n=4 per group) in the presence or absence of NK cells (CD45.2). Bone marrow
was obtained from recipients 30 hours after transplant and the percentage of both CD45.1/2 lineage
negative cells and LKS cells was measured by flow cytometry analysis. The addition of NK cells to the
donor cell preparations did not affect homing of progenitor cells, lineage negative as well as LKS donor
cells were quantified, Two-tailed Student’s t-test was used to assess statistical significance, ns: not
significant (p ≥ 0.05). Results are shown by normalization of the percentages of recovered cells in the LKS+
NK group to the LKS injected group.

Supplemental Information Page 3

A

Pre- Post IgG- Post NK-

Ter119/CD19/CD4/CD8

depletion depletion depletion

CD3
CD3

0.98 1.19 0.039

NK1.1

B
Blood Bone marrow Spleen
125
% CD45.1 + cells

100
75

50
25
0
6.25 1.25 0.25 6.25 1.25 0.25 6.25 1.25 0.25 6.25 1.25 0.25 6.25 1.25 0.25 6.25 1.25 0.25

IgG control NK1.1 depleted IgG control NK1.1 depleted IgG control NK1.1 depleted

T cells Myeloid B cells

Figure S3. NK cell depletion from murine BM grafts improves hematopoietic recovery of recipient
mice. Related to figure 3. (A) Flow cytometry analysis of BM donor samples prior to depletion (pre-
depletion), IgG control depleted (post IgG-depletion), and NK cell depleted (post NK-depletion). Upper dot
plots indicate Ter119/CD19/CD4/CD8 staining versus CD3 staining. Black box indicates gating used for
further analysis shown in lower panels. Lower dot plots show CD3 and NK1.1 staining. Orange box
indicates percentage of NK cells (CD3-NK1.1+) from alive cells. (B) Lineage contribution of CD45.1+ donor
cells to blood formation in recipient mice. Y axes indicate the percentage of CD45.1+ cells to each lineage. X
axes indicate the different groups based on IgG control and NK1.1 depletion. Cell doses used for
transplantation are indicated as 6.25 (6.25 x 106 cells), 1.25 (1.25 x 106 cells) and 0.25 (0.25 x 106 cells).
Gray boxes indicate the percentage of T cells, red boxes the percentage of myeloid cells, and blue boxes the
percentage of B cells. Each column represents average values and standard deviation for at least 5
mice/group. Supplemental Information Page 4
 Total BM
CD3 CD3 +
+ IgG CD56

MACS MACS

Negative fraction Positive fraction Negative fraction Positive fraction

after CD3 + IgG after CD3 + IgG after CD3 + CD56 after CD3 + CD56
depletion depletion depletion depletion
SSC

CD45
CD19

CD3
SSC

CD56

Figure S4. Human bone marrow fractionation. Related to figure 4. Upper scheme: graphical
representation of the procedure. BM cells were fractionated based on CD3 and IgG expression or CD3 and
CD56 expression. After MACS separation negative and positive fractions were recovered. Lower flow
cytometric plots: Separated BM populations were stained using antibodies against CD45, CD19, CD3, and
CD56. Red arrow points to the efficiently deleted NK cells.

Supplemental Information Page 5

 A B NKG2A NKG2D Ly49D C
14
ns
ns ns ns
p=0.0078
60 80 50

% of NK cell activation
12

(% of NK1.1+ cells)
10 50 40
60
% NK cells

(CD107a)
8 40
30
6 30 40
20
4 20
20 10
2 10
0 0 0 0
WT KO WT KO WT KO WT KO WT KO

D p=0.048 E HSC + IL-2

p=0.002
1000 100%
# alive HSCs

500
50%
0
HSC: alone +WT +Cebpg KO 0%
NK cells 1 2 3 4 5 6 7 8 9 10

HSC + WT NK + IL2 HSC + Cebpg KO NK + IL2

100% 100%
T cells
Myel cells
50% 50%
B cells

0% 0%
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
Figure S5. Characterization and function of Cebpg KO NK cells in comparison to WT NK cells.
Related to figure 5. (A) Percentage of NK cells determined by flow cytometry analysis in WT and Cebpg
KO spleen cells. NK cells were determined by expression of NK1.1 antigen in the absence of Ter119, CD4,
CD8, CD19 and CD3. (B) Expression of the receptors NKG2A, NKG2D and Ly49H in WT and Cebpg KO
NK cells determined by flow cytometry (n=4 per group). Y axes indicate the expression of each receptor
among NK1.1+ gated cells. (C) NK cell cytotoxicity against the murine YAC-1 MHC-I-deficient lymphoma
cell line determined by the detection of CD107a by flow cytometry after five hours of a 10:1 ratio of
NK:YAC-1 co-culture. Y axis illustrates the percentage (%) of CD107a among NK1.1+ gated cells. (D)
Number of HSCs assessed by FACS. Y axis indicates number of alive Hoescht 33258- HSCs after overnight
incubation. X axis indicates culture conditions. (E) Flow cytometry analysis of peripheral blood samples
from recipient mice 16 weeks post-transplant. Donor CD45.1/2 cells were analyzed for expression of T
(CD3, grey box), myeloid (Gr-1 and CD11b, red box) or B (B220, blue box) cell lineage markers. Y axes
indicates percentage of the distinct populations. X axes indicate individual animals: 1-10 transplanted with
HSC alone, 11-15 transplanted with HSC co-cultured with WT NK cells, 16-27 transplanted with HSC co-
cultured with Cepbg KO NK cells, all co-cultures were kept in media with IL-2. Two-tailed Student’s t-test
was used to assess statistical significance (p values are indicated, ns: not significant).
Supplemental Information Page 6
Figueiredo-Pontes et al NK CELLS REGULATE HSC FUNCTION

TABLE S1. List of genes belonging to the NK cell signature. Related to Figure 6.

TABLE S2. Differential pathway enrichment analysis in WT and Cebpg KO NK cells. Related to Figure 6.

Combined
Index Name P-value Z-score
Score
1 toll like receptor signaling pathway 0.002136 -1.90 3.08
2 apoptosis 0.003123 -1.85 3.00
3 b cell receptor signaling pathway 0.003992 -1.75 2.84
4 vegf signaling pathway 0.01860 -1.82 1.05
5 aminoacyl trna biosynthesis 0.02612 -1.55 0.89
6 pancreatic cancer 0.02696 -1.69 0.97
7 ubiquitin mediated proteolysis 0.02886 -1.28 0.73
8 chronic myeloid leukemia 0.03096 -1.61 0.93
9 c21 steroid hormone metabolism 0.03397 0.75 -0.43
10 cytokine cytokine receptor interaction 0.04043 -1.53 0.87
11 MAPK signaling pathway 0,04184 -1.54 0,87

Results of KEGG pathway analysis of WT versus Cebpg KO NK cells with the threshold of p<0.05.

Supplemental Information Page 7

Figueiredo-Pontes et al NK CELLS REGULATE HSC FUNCTION

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Co-cultures with OP9 stromal cells

Co-cultures of HSCs (lin- c-Kit+ Sca-1+ CD48- CD150+) and NK cells (Ter 119- CD19- CD8- CD4- CD3- NK1.1+)
in different ratios were performed using serum-free stem cell medium and cytokines (100 ng/mL SCF, 100
ng/mL FLT3-ligand, 10 ng/mL IL-3, 10 ng/mL IL-6, 25 ng/mL TPO, murine IL-2 1000 U/mL) over a confluent
layer of mitomycin C treated OP9 stromal cells. 4 days after culture cells were stained for HSC markers (lin- c-
Kit+ Sca-1+ CD48- CD150+), enumerated by flow cytometry analysis, and colonies were visualized by
microscopy.

Murine colony-forming unit (CFU) assays

Co-cultures of HSCs and NK cells were performed using StemSpan serum-free stem cell medium and cytokines
(100 ng/mL SCF, 100 ng/mL FLT3-ligand, 10 ng/mL IL-3, 10 ng/mL IL-6, 25 ng/mL TPO, IL-2 1000 U/mL) in
96-well round bottom plates overnight. When indicated, aforementioned media was supplemented with 10
ug/mL neutralizing anti-IFN gamma monoclonal Antibody (clone XMG1.2, eBioscience) or Rat IgG1 kappa
Isotype Control (eBRG1, eBioscience). After overnight incubation, cells were plated in methylcellulose-based
medium MethoCult GF M3434 (Stemcell Technologies) supplemented with murine IL-2 1000 U/mL. 100
murine HSCs alone or 100 HSCs co-cultured with 1000 or 5000 NK cells were plated in duplicates in 12-well
plate. Number of colonies was assessed 10 days after plating.

Human CFU assays

Human CD34+ cells and NK cells (CD19- CD8- CD4- CD3- CD56+) were isolated from adult bone marrow (BM)
obtained from femoral heads removed during hip replacement surgeries. First, mononuclear cells were separated
by density gradient centrifugation using Ficoll-Paque PREMIUM (GE Healthcare). One part of the mononuclear
cell suspension was frozen and later human NK cells were sorted from thawed material by using BD
FACSAriaIIu. Another part was immediately used to obtain CD34+ cells by magnetic separation using human
CD34+ MicroBead kit (Miltenyi Biotec) and autoMACS Separator (Miltenyi Biotec). CD34+ cells were frozen as
well. Thawed human CD34+ cells were cultured alone or with NK cells from the same patient (ratio 1:10) in
serum-free stem cell medium and human cytokines (100 ng/mL SCF, 50 ng/mL G-CSF, 100 ng/mL FLT3-
ligand, 25 ng/mL IL-3, 20 ng/mL TPO, IL-2 1000 U/mL) in 96-well round bottom plate overnight. After
overnight incubation, cell suspensions of 500 or 1000 CD34 + cells alone or co-cultured with 5000 or 10000 NK
cells, respectively, were plated in duplicates in 12-well plate and number of colonies was assessed 10 days after
plating. CFU assays were performed using MethoCult H4535 (Stemcell Technologies) supplemented with
human IL-2 1000 U/mL (Gibco).

Gene expression analysis

Total RNA was isolated from NK cells sorted from WT and Cebpg KO murine spleens cultured in RPMI with
10% FBS overnight in the presence of 1000 U/ml IL-2 (PeproTech) or vehicle. RNA extraction was performed
using RNeasy Plus Mini Kit (Qiagen). RNA integrity was analyzed by Agilent Bioanalyzer 2100 (Agilent), and
only samples with intact RNA profiles were used for expression profiling (RIN > 7). Four biological replicates
were used for each phenotype and treatment. 600 pg RNA was amplified with PicoSL WTASystem V2
(NuGEN), labeled with Encore Biotin IL Module (NuGEN) and 750 ng of labeled RNA was hybridized on
MouseRef-8 v2.0 Expression BeadChip (Illumina) according to the manufacturer procedure. Raw data were
processed using the beadarray package of Bioconductor and analyzed as described previously(Melenovsky et al.,
2011). Gene set enrichment analysis (GSEA) was performed using the Enrichr gene analysis tool
(http://amp.pharm.mssm.edu/Enrichr)(Chen et al., 2013). Microarray data were deposited in ArrayExpress
(http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5604.

Cytokine assay
A total of 250.000 WT or Cebpg KO purified NK cells were cultured in RPMI1640 supplemented with 10% FBS
and 1000 U/mL of IL-2 for 24 hours. Then, supernatants were collected, and IL2, IL4, IL6, IL10, IL17A, TNF
and IFN cytokine levels were measured by flow cytometry analysis using Cytometric Bead Array technology
(BD Biosciences) according to manufacturer’s instructions.

Murine BM transplantation
1500 HSCs, defined as lin- c-Kit+ Sca-1+ CD48- CD150+ cells, were sorted from mice (CD45.1/2) and cultured
with or without IL-2 and/or NK cells (CD3- NK1.1+) in a ratio of 105 NK cells to 1.5 x 103 HSCs per mouse. The
100:1.5 NK:HSC ratio was chosen to roughly mimic the proportions of these two cell subtypes in a normal BM
(approximately 1% of NK cells and 0.01% of HSCs). Then, the content of individual wells was mixed together
with 0.5 × 106 BM cells as a support (CD45.1) and injected through the tail vein into lethally irradiated (6 Gy)

Supplemental Information Page 8

Figueiredo-Pontes et al NK CELLS REGULATE HSC FUNCTION

CD45.1 recipients (3 independent experiments were performed) For engraftment analysis, cells were stained
with anti-CD45.1 and CD45.2 antibodies to distinguish donor-derived cells from the host cells, as well as with
lineage-specific antibodies Mac1, Gr1, B220 and CD3 to identify myeloid, B and T lineages, respectively.
Engraftment analysis was performed after 9 and 16 weeks of transplant.
For murine limiting dilution assays, total BM cells (CD45.1) were submitted to NK1.1 or IgG depletion and then
different numbers injected into lethally irradiated (6 Gy) CD45.2 recipients (n=6 or 11 per group, 2
experiments). Peripheral blood was collected from each mouse at 8 and 16 weeks, and BM and spleen were
obtained at 16 weeks for final chimerism analysis. For this assay, a recipient mouse was considered positive if
>0.1% CD45.1+ cells were detected and three lineages were reconstituted (>1% of CD3+, Gr1+Mac1+ or B220+
cells from CD45.1+ cell population).

NK depletion from murine and human BM

After red blood cells lyse, cells from murine whole BM were stained with 0.1 μg per 106 cells (in a 1:50 dilution)
of anti-mouse IgG2a (MOPC-173) or NK1.1 (PK136) biotinylated antibodies (Biolegend) for 30 minutes,
washed and stained with anti-biotin Microbeads Ultrapure as recommended by manufacturer, and then submitted
to immunomagnetic depletion using an auto MACS separator (Miltenyi Biotec). After depletion, cell suspensions
were prepared in sterile PBS for mice injection. Depletion efficiency was tested in parallel in all experiments by
staining samples before depletion and after IgG or NK depletion with other lineage markers (Ter119, CD19,
CD4, CD8), CD3 and NK1.1. NK cells were gated as viable, lineage negative, CD3 negative and NK1.1 high. For
human NK cells depletion, BM was obtained from healthy volunteers after informed consent and in accordance
to Institutional policies (1st Department of Medicine - Department of Haematology, First Faculty of Medicine,
Charles University in Prague and General University Hospital, Prague, Czech Republic). Fresh BM cells were
submitted to mononuclear cell isolation by Ficoll Hypaque density gradient, washed with sterile PBS/20% FBS,
stained with 0.25 μg per 106 cells of anti-human biotinylated CD3 (MEM-57) and IgG2a (MOPC-173) or CD56
(MEM-188), and then taken to immunomagnetic separation with streptavidin beads. T-cell depletion with CD3
was performed in both IgG and CD56 depleted experimental arms in order to avoid GVHD and improve survival
of the recipient mice. In human experiments, CD56 depletion efficiency was tested by staining pre-depletion and
post-depletion samples with CD45, CD19, CD3, CD56 (Figure S4). Serial cell dilutions were prepared in sterile
PBS for injection into mice.

Xenotransplantation
NK cell-depleted (CD56) or non-depleted (IgG) human grafts were transplanted into sublethally irradiated (200
cGy) NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice at different doses. The percentages of human CD45 + cells in
the murine blood (8 and 14 weeks) and BM (14 weeks) after transplantation were determined.

Mobilized blood transplantation and IFN treatment

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were transplanted with 2 x 10 6 human mobilized blood cells
containing an equivalent of 3.6 x 105 CD34+ cells. Mice were treated at day 0, 3, 7, 10, and 14 after
transplantation with 100 g murine anti-human IFN neutralizing monoclonal antibody (clone NIB42, Exbio,
Czech Republic) or murine isotype control IgG1 monoclonal antibody (clone PPV-06, Exbio, Czech Republic).
Engraftment was determined at day 17 after transplantation by assessing the percentage of human CD45+ and
human CD34+ cells in the BM.

Supplemental Information Page 9

Figueiredo-Pontes et al NK CELLS REGULATE HSC FUNCTION

SUPPLEMENTAL REFERENCES

Chen, E.Y., Tan, C.M., Kou, Y., Duan, Q., Wang, Z., Meirelles, G.V., Clark, N.R., and Ma'ayan, A. (2013).
Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC bioinformatics 14, 128.
Melenovsky, V., Benes, J., Skaroupkova, P., Sedmera, D., Strnad, H., Kolar, M., Vlcek, C., Petrak, J., Benes, J.,
Jr., Papousek, F., et al. (2011). Metabolic characterization of volume overload heart failure due to aorto-caval
fistula in rats. Molecular and cellular biochemistry 354, 83-96.

Supplemental Information Page 10