Abstract
Objectives
Ruxolitinib, a Janus kinase (JAK) 1/2 inhibitor, demonstrates efficacy
for treating steroid‐resistant acute graft‐versus‐host disease
(SR‐aGVHD) following allogeneic stem cell transplantation (allo‐HSCT).
Myeloid‐derived suppressor cells (MDSCs) have a protective effect on
aGVHD via suppressing T cell function. However, the precise features
and mechanism of JAK inhibitor‐mediated immune modulation on MDSCs
subsets remain poorly understood.
Methods
A total of 74 SR‐aGVHD patients treated with allo‐HSCT and ruxolitinib
were enrolled in the present study. The alterations of MDSC and
regulatory T cell (Treg) populations were monitored during ruxolitinib
treatment in responders and nonresponders. A mouse model of aGVHD was
used to evaluate the immunosuppressive activity of MDSCs and related
signalling pathways in response to ruxolitinib administration in vivo
and in vitro.
Results
Patients with SR‐aGVHD who received ruxolitinib treatment achieved
satisfactory outcomes. Elevation proportions of MDSCs before treatment,
especially polymorphonuclear‐MDSCs (PMN‐MDSCs) were better to reflect
the response to ruxolitinib than those in Tregs. In the mouse model of
aGVHD, the administration of ruxolitinib resulted in the expansion and
functional enhancement of PMN‐MDSCs and the effects could be partially
reversed by an anti‐Gr‐1 antibody in vivo. Ruxolitinib treatment
significantly elevated the suppressive function of PMN‐MDSCs through
reactive oxygen species (ROS) production by Nox2 upregulation as well
as bypassing the activated MAPK/NF‐κB signalling pathway. Additionally,
ex vivo experiments demonstrated that ruxolitinib prevented the
differentiation of mature myeloid cells and promoted the accumulation
of MDSCs by inhibiting STAT5.
Conclusions
Ruxolitinib enhances PMN‐MDSCs functions through JAK/STAT and
ROS‐MAPK/NF‐κB signalling pathways. Monitoring frequencies and
functions of MDSCs can help evaluate treatment responses to
ruxolitinib.
Keywords: acute graft‐versus‐host disease, JAK/STAT pathway,
myeloid‐derived suppressor cells, ROS
__________________________________________________________________
Our results provide rationale for molecular modification of
myeloid‐derived suppressor cells to augment the efficacy of ruxolitinib
in patients with acute graft‐versus‐host disease, which may be
applicable to other pathologies including autoimmunity, chronic
inflammation and cancer.
graphic file with name CTI2-12-e1441-g007.jpg
Introduction
Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is a
well‐established and effective therapeutic intervention for
haematopoietic malignant and nonmalignant diseases. However, allo‐HSCT
is associated with severe complications, including acute
graft‐versus‐host disease (aGVHD).[50] ^1 , [51]^2 aGVHD involves
aberrant activation of donor‐derived naïve T cells, which recognise
host antigens and subsequently attack target organs, including the
skin, liver and gastrointestinal tract (GI).[52] ^3 Although
substantial progress has been made in the development of prophylactic
and immunosuppressive therapies for aGVHD in recent decades, 30–50% of
patients undergoing allo‐HSCT develop aGVHD, which is associated with
poor prognosis.[53] ^4 Further, approximately half of the patients
treated with glucocorticoid as first‐line therapy for aGVHD develop
steroid‐refractory acute GVHD (SR‐aGVHD), which accounts for
significant mortality.[54] ^5 Ruxolitinib, a Janus kinase (JAK) 1/2
inhibitor, was recently approved for treating SR‐aGVHD as it can impair
the differentiation of CD4^+ T cells into IFN‐γ‐ and IL‐17A‐producing
cells and increase the proportion of Foxp3^+ regulatory T cells
(Tregs).[55] ^6 , [56]^7 However, the timing of ruxolitinib therapy
standard indicator for evaluating treatment response to ruxolitinib is
still largely unknown.
Myeloid‐derived suppressor cells (MDSCs) are a heterogeneous population
of immunosuppressive myeloid cells that play a beneficial role in
transplantation by suppressing alloreactive T cell responses, thereby
exerting protective effects against the development of typical aGVHD
and reducing transplantation‐related deaths.[57] ^8 , [58]^9 MDSCs are
defined as CD11b^+Gr‐1^+ and Lin^−CD11b^+HLA‐DR^− cells in mice and
humans, respectively. MDSCs comprise two subsets characterised by
distinct morphological features: polymorphonuclear (PMN) and monocytic
(M)‐MDSCs.[59] ^10 PMN‐MDSCs express high levels of reactive oxygen
species (ROS) owing to the activity of nicotinamide adenine
dinucleotide phosphate (NADPH), whereas M‐MDSCs contain nitric oxide
(NO) and arginase‐1 (Arg‐1) because of the upregulation of signal
transducer and activator of transcription 1 (STAT1) and inducible NO
synthase (NOS). Both subsets exert potent suppressive effects on T
cells and induce immunological tolerance following transplantation.[60]
^11 , [61]^12
Myeloid‐derived suppressor cells are induced by various inflammatory
factors, including granulocyte/macrophage colony‐stimulating factor
(GM‐CSF), granulocyte CSF, vascular endothelial growth factor,
interleukin‐6 (IL‐6), interferon‐γ (IFN‐γ), tumor necrosis factor‐α
(TNF‐α), IL‐1β and toll‐like receptor ligands.[62] ^13 Most of these
factors trigger signalling pathways in MDSCs that converge on JAK
protein family members and STAT3/5, which are involved in the expansion
and differentiation of MDSCs in cancer.[63] ^14 Ablation of STAT3
expression using conditional knockout mice or selective STAT3
inhibitors markedly reduces the expansion of MDSCs and increases T cell
responses in tumor‐bearing mice.[64] ^15 Furthermore, persistent
activation of STAT3 blocks the differentiation of myeloid progenitor
cells by upregulating S100A8/9 and increases their survival by inducing
the expression of B‐cell lymphoma xl (Bcl‐xl) and cyclin D1 in the
tumor microenvironment (TME).[65] ^16 , [66]^17 , [67]^18 Meanwhile,
STAT5 also contributes to the proliferation and survival of immature
myeloid cells and prevents their differentiation into mature cells.[68]
^19 However, the role of JAK/STAT signalling of MDSCs in the
pathogenesis of aGVHD has not been documented.
In this study, we evaluated the biological functions of MDSCs on
therapeutic process of JAK1/2 inhibition interfering with aGVHD after
allo‐HSCT. The administration of ruxolitinib as second‐line treatment
of aGVHD may enhance the immunosuppressive function of MDSCs and block
their differentiation into mature cells. PMN‐MDSCs were found to
increase the levels of ROS in a MAPK/NF‐κB‐dependent manner.
Considering the lack of standard indicators for evaluating treatment
responses in patients with aGVHD,[69] ^20 we demonstrated that MDSC
status can predict treatment response to ruxolitinib therapy in
patients with SR‐aGVHD. These results provide further evidence that
MDSCs are the therapeutic targets of ruxolitinib and the evaluation of
MDSC population and function may have utility in patient selection for
developing individualised treatment strategies.
Results
Outcomes following ruxolitinib treatment in patients with SR‐aGVHD
We enrolled 74 patients who received ruxolitinib as second‐line therapy
for SR‐aGVHD. Patient characteristics are summarised in Supplementary
table [70]1. The median age of patients was 35 years (range,
8–63 years), and 48.6% of them were men. Of these, 7 patients (9.5%)
had grade II aGVHD, 33 (44.6%) had grade III aGVHD, and 34 (45.9%) had
grade IV aGVHD. The overall response rate (ORR) at day 28 was 68.9%
(51/74), with 29.7% of patients achieving CR after a median of 8 days
(range, 1–40 days). The proportion of patients with durable ORR at day
56 was 63.5% (47/74), with 47.3% of patients achieving CR (35/74;
Figure [71]1a). Over a median follow‐up period of 298 (7–1495) days,
there was a significantly increased 6‐month OS in aGVHD patients with
responders (68.6% ± 6.5%) compared with nonresponders (21.7% ± 8.6%;
P < 0.001, Figure [72]1b).
Figure 1.
Figure 1
[73]Open in a new tab
Ruxolitinib treatment improved the overall response of patients with
SR‐aGVHD after allo‐HSCT along with alterations of myeloid‐derived
suppressor cells (MDSCs). (a) The overall response rate (ORR) at day 28
and day 56 in patients with SR‐aGVHD who received ruxolitinib treatment
after allo‐HSCT (n = 74). (b) The overall survival of patients with
ruxolitinib for aGVHD according to the response of ruxolitinib
(Responders who obtained PR or above, n = 51; Nonresponders who failed
treatment, n = 23). (c) The percentages of MDSCs (CD11b^+HLA‐DR^−),
PMN‐MDSCs (CD11b^+HLA‐DR^−CD14^−CD15^+), M‐MDSCs
(CD11b^+HLA‐DR^−CD14^+CD15^−), Tregs (CD4^+CD25^+Foxp3^+) from PB
samples in responders (n = 7) and nonreponders (n = 6) before and
within 7 days, 14 days after starting ruxolitinib. (d, e) Correlation
analysis of the MDSCs and PMN‐MDSCs proportions before ruxolitinib
administration compared with response to ruxolitinib treatment. Each
dot represents an independent core. The response to ruxolitinib
included response (CR + PR) and nonresponse (0 = nonresponse,
1 = response, respectively). r and P‐values were calculated using
Spearman's correlation test. *P < 0.05, **P < 0.01.
Cytopenias are common complications of ruxolitinib treatment. The
incidence of grade III or IV anaemia, neutropenia and thrombocytopenia
was 17.6% (13/74), 17.6% (13/74) and 14.9% (11/74), respectively.
Bacterial, viral and fungal infections were common in patients with
SR‐aGVHD receiving ruxolitinib treatment and included cytomegalovirus
(CMV) viremia (58.1%), Epstein–Barr viremia (4.05%), fungal infections
(5%) and bacterial/viral infections (10.8%). These findings highlight
the importance of infectious surveillance in patients receiving
ruxolitinib treatment (Supplementary table [74]2). Relapse of the
underlying malignancy occurred in 18.9% (14/74) of patients during the
follow‐up period.
The alterations of MDSCs, especially PMN‐MDSCs, were favorable indicators to
evaluate the treatment response to ruxolitinib in aGVHD patients
Ruxolitinib is a promising treatment option for SR‐aGVHD and can
achieve favorable ORR and OS without serious side effects. However,
there are no consensual indicators to reflect treatment responses. To
evaluate the role of circulating immunoregulatory cells obtained from
PB samples, alterations in the levels of MDSCs and Tregs were monitored
in patients with aGVHD before and after ruxolitinib treatment,
including 7 responders and 6 nonresponders. As shown in Figure [75]1c,
the number of MDSCs (CD11b^+HLA‐DR^− cells) significantly increased at
day 7 and declined at day 14 in responders; however, the numbers were
constantly increasing in nonresponders after ruxolitinib treatment.
More importantly, we assessed two major MDSC subpopulations:
CD11b^+HLA‐DR^− CD15^+CD14^− PMN‐MDSCs and CD11b^+HLA‐DR^− CD15^−CD14^+
M‐MDSCs. There was a similar tendency in PMN‐MDSCs. However, M‐MDSCs
were not related to the treatment response of ruxolitinib. Patients who
displayed a significant response to treatment had statistically higher
frequencies of MDSCs and PMN‐MDSCs before the administration of
ruxolitinib than patients who showed treatment failure (P < 0.05).
Correlation analysis showed a significant positive correlation between
the proportions of MDSCs and PMN‐MDSCs before ruxolitinib treatment and
treatment response (P = 0.046 and 0.047, respectively, Figure [76]1d
and e). A recent study suggested that Tregs play a critical role in
allo‐HSCT, which was closely associated with the severity of aGVHD.[77]
^21 We further analysed the percentage of Tregs during ruxolitinib
treatment. There was a higher frequency of Tregs in responders than in
nonresponders within day 7 and day 14 after starting ruxolitinib
treatment; however, this difference was not significant
(Figure [78]1c). These data indicate that alterations in circulating
MDSCs, especially PMN‐MDSCs, were more sensitive than those in Tregs
and may predict and reflect the response of patients with SR‐aGVHD to
ruxolitinib. Nevertheless, we need large‐scale cohort studies to verify
this conclusion.
Ruxolitinib treatment alleviated aGVHD symptoms and induced the expansion of
MDSCs, particularly PMN‐MDSCs
To determine the effects of ruxolitinib on established parameters of
aGVHD, we employed a major histocompatibility complex (MHC)‐mismatched
(C57BL/6 to BALB/c) murine model of aGVHD. Ruxolitinib or the vehicle
was administered by oral gavage from day 0 to day 30 (Figure [79]2a).
Ruxolitinib treatment resulted in significant improvements in survival
as well as increased the body weight and clinical scores of mice after
transplantation compared with the vehicle (Figure [80]2b–d). Mice that
received BM only developed no signs of aGVHD and had a survival rate of
100% until the endpoint. Histological analysis of liver, intestine and
colon tissues from day 7 revealed decreased pathological damage and
inflammatory cell infiltration in mice treated with ruxolitinib
(Figure [81]2e). No apparent cytopenias were observed upon blood
routine test in ruxolitinib‐treated mice (Supplementary
figure [82]1a–d).
Figure 2.
Figure 2
[83]Open in a new tab
Ruxolitinib treatment reduced aGVHD severity and promoted
myeloid‐derived suppressor cells (MDSCs) expansion. Lethally irradiated
BALB/c mice were transplanted with 1 × 10^7 BM cells with or without
3 × 10^7 splenic cells from C57BL/6 mice. Recipient mice received
vehicle or ruxolitinib (30 mg/kg) by oral gavage twice a day after
transplantation. (a) Schematic representation of the experimental
procedure. The overall survival (b), weight variation (c) and clinical
GVHD score (d) were shown in each group (BM‐only group, n = 6; Vehicle
group, n = 12; Ruxolitinib group, n = 12). (e) Representative
haematoxylin & eosin‐stained sections of the liver, intestine and colon
from vehicle and ruxolitinib‐treated mice at day 7 after
transplantation (scale bar, 100 μm). The arrows were used to indicate
the representative areas. (f–i) The percentages and absolute numbers of
donor‐derived splenic MDSCs (CD11b^+Gr‐1^+), PMN‐MDSCs
(CD11b^+Ly6G^+Ly6C^lo) and M‐MDSC (CD11b^+Ly6G^−Ly6C^hi) were measured
with flow cytometry in vehicle treatment and ruxolitinib treatment
groups at day 7 after transplantation (n = 4 or 5 per group). (j) The
morphology of splenic‐derived PMN‐MDSCs isolated from vehicle‐ and
ruxolitinib‐treated mice at day 7 after transplantation, which were
visualised by Wright–Giemsa staining under light microscope (scale bar,
10 μm). *P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001.
To determine the effect of ruxolitinib on T cell fate in mice with
aGVHD, we performed donor‐derived T cell profiling. The proportions of
activated CD4^+CD69^+ T and IFN‐γ‐producing CD4^+ Th1 cells were
significantly lower and the frequencies and absolute numbers of
CD4^+Foxp3^+ Tregs in the spleen on day 7 were significantly higher in
mice treated with ruxolitinib than in those treated with the vehicle,
indicating attenuation of the inflammatory response in aGVHD
(Supplementary figure [84]2a–d). In addition, ruxolitinib treatment
considerably suppressed the production of proinflammatory cytokines,
including IFN‐γ, TNF‐α, MCP‐1, IL‐12p70, IL‐6, IL‐17A and GM‐CSF and
significantly increased the serum levels of anti‐inflammatory IL‐10
(Supplementary figure [85]2e). Consistent with the findings of a
previous investigation,[86] ^6 these results demonstrate that the
protective effect of ruxolitinib is mediated by reducing T cell
pathogenicity and inflammatory cytokine production during the
development of aGVHD.
Our initial analysis demonstrated that the expansion of MDSCs,
particularly PMN‐MDSCs, was closely correlated with treatment response
to ruxolitinib during aGVHD progression. These results are consistent
with those of a previous study, reporting that the JAK/STAT pathway
plays an important role in the recruitment of MDSCs to inflammatory
sites.[87] ^22 We therefore hypothesised that ruxolitinib affects MDSC
accumulation in aGVHD. As shown in Figure [88]2f–i, the proportions and
absolute numbers of MDSCs (CD11b^+Gr‐1^+) were significantly increased
in the spleen of mice following treatment with ruxolitinib than those
with vehicle treatment, particularly at day 7 after transplantation.
Moreover, we sought to delineate the alterations in splenic PMN‐MDSCs
(CD11b^+Ly6G^+Ly6C^lo) and M‐MDSCs (CD11b^+Ly6G^‐Ly6C^hi).
Interestingly, the proportions and absolute numbers of PMN‐MDSCs in the
spleen of ruxolitinib‐treated mice were significantly higher than those
in the spleen of vehicle‐treated mice at day 7 and 14 after
transplantation. Variation in the proportion of M‐MDSCs was less
affected by ruxolitinib treatment than that in the proportion of
PMN‐MDSCs.
Ruxolitinib enhanced the suppressive activity of MDSCs in aGVHD
We next evaluated the effect of ruxolitinib on the suppressive function
of MDSCs and related subsets in aGVHD. MDSCs, PMN‐MDSCs and M‐MDSCs
were isolated from splenocytes of vehicle and ruxolitinib group and
cocultured with T cells purified from the spleen of wild‐type C57BL/6
at different ratios (MDSC: T = 0:1 to 1:2) for 3 days stimulated by
anti‐CD3/CD28 beads. MDSCs isolated from vehicle‐treated mice showed
reduced capacity to block T cell proliferation compared with those
isolated from ruxolitinib‐treated mice. The immunosuppressive function
of PMN‐MDSCs was significantly enhanced and that of M‐MDSCs was
impaired in the ruxolitinib group compared with those in the vehicle
group (Figure [89]3a–c). We further investigated the morphology of
PMN‐MDSCs isolated from the two groups under light microscope.
PMN‐MDSCs isolated from ruxolitinib‐treated mice had larger cell
volumes and richer cytoplasm than those isolated from vehicle‐treated
mice, which may contribute to the increased suppressive effects of T
cells (Figure [90]2j).
Figure 3.
Figure 3
[91]Open in a new tab
Ruxolitinib treatment enhanced immunosuppressive function of MDSCs,
especially PMN‐MDSCs in aGVHD mice. Depletion of MDSCs exacerbated
aGVHD lethality in ruxolitinib‐treated mice. (a–c) CFSE‐labelled CD3+ T
cells (1 × 10^5 per well) from wild‐type C57BL/6 spleen were stimulated
with CD3/28 beads and cocultured with different ratios of purified
splenic MDSCs, PMN‐MDSCs and M‐MDSCs isolated from vehicle‐ and
ruxolitinib‐treated mice at day 7 after transplantation for 72 h.
Proliferation of CFSE‐labelled CD3+ T cells was measured with flow
cytometry (Vehicle group, n = 3; Ruxolitinib group, n = 3). (d) The
helper T cell‐related cytokines were detected in supernatants harvest
from the above coculture system (n = 3). (e,f) aGVHD mouse models were
built as described previously. Two hundred microgram anti‐Gr‐1 antibody
was injected intraperitoneally into recipient mice with ruxolitinib
treatment to deplete MDSCs as Gr‐1 depletion group from day 5 to day 29
every other day after transplantation. The overall survival and weight
ratio were exhibited in each group (Vehicle group, n = 6; Ruxolitinib
group, n = 6; Gr‐1 depletion group, n = 5). Data are expressed as
mean ± standard error (SE) and from three independent experiments.
**P < 0.01, ***P < 0.001.
Levels of helper T (Th) cell‐related cytokines in the supernatants
derived from the PMN‐MDSCs suppression assay were examined using a
multiplex kit. Compared with vehicle group, supernatants of PMN‐MDSCs
from ruxolitinib group which cocultured with T cells exhibited apparent
defects in the expression of proinflammatory cytokines such as Th1
cytokines‐IFN‐γ, TNF‐α and Th17 cytokines‐IL‐17. By contrast, the
levels of anti‐inflammatory cytokines such as Treg cytokines (IL‐10)
were increased in the supernatants of cultures containing PMN‐MDSCs
isolated from the ruxolitinib group (Figure [92]3d). Overall, these
results indicate that ruxolitinib alters the distribution of MDSCs
towards PMN‐MDSCs, which modulate Th cell balance and exert
immunosuppressive effects by inhibiting the proliferation of T cells.
MDSC depletion weakened the therapeutic efficacy of ruxolitinib in aGVHD
As we demonstrated the effect of ruxolitinib in maintaining MDSC
function, we next evaluated the protective effect of MDSCs in GVHD
following ruxolitinib treatment. CD11b^+Gr‐1^+ MDSCs in the BM, spleen
and blood can reportedly be depleted using an anti‐Gr1 antibody.[93]
^23 Accordingly, we injected 200 μg of anti‐Gr‐1 antibodies into
recipient mice treated with ruxolitinib every other day from day 5 to
day 29 after transplantation, and flow cytometry revealed almost
complete depletion of MDSCs (Supplementary figure [94]2f). As shown in
Figure [95]3e and f, compared with mice receiving ruxolitinib alone,
Gr‐1 depletion significantly decreased survival and accelerated weight
loss caused by aGVHD in the ruxolitinib‐treated mice. This indicates
that MDSCs are required at the stage of post‐transplantation for
ruxolitinib treatment to suppress aGVHD.
Transcription signatures of PMN‐MDSCs in aGVHD mice with or without
ruxolitinib treatment
To determine the mechanisms underlying the increased suppressive
effects of PMN‐MDSCs in ruxolitinib‐treated mice with aGVHD, PMN‐MDSCs
were isolated from mice with aGVHD treated with or without ruxolitinib
at day 7 after transplantation. RNA‐seq was then used to evaluate the
transcription signatures of PMN‐MDSCs. Pathway enrichment analysis was
performed using gene ontology (GO), Kyoto Encyclopedia of Genes and
Genomes pathway database analysis and gene set enrichment analysis
(GSEA). In PMN‐MDSCs isolated from ruxolitinib‐treated mice, GSEA
analysis revealed that oxidative phosphorylation, Notch signalling and
TGF‐β signalling were upregulated, whereas IFN‐γ and IL6‐JAK/STAT3
pathways were downregulated (Figure [96]4a). GO analysis indicated that
differentially upregulated genes in the ruxolitinib group were
particularly enriched in the negative regulation of apoptosis, ROS
metabolic processes, NF‐κB signalling and MAPK activity. By contrast,
PMN‐MDSCs from the vehicle group exerted impaired suppressive effects
on T cells by promoting T cell activation, cellular responses to IFN‐γ
and STAT phosphorylation (Figure [97]4b). Genes with elevated
expression in PMN‐MDSCs from the ruxolitinib group were enriched for M2
anti‐inflammatory signatures compared with those from the vehicle group
(Figure [98]4c). qRT–PCR demonstrated increased expression of M2
signatures, including genes related to ROS production (Nox2, Hif1α and
Ho‐1) and immunosuppressive functions (Il10 and Tgfb1). No significant
difference in mRNA expression of Arg1, Nos2 or Cox2 was observed
between the two groups (Figure [99]4d).
Figure 4.
Figure 4
[100]Open in a new tab
Transcriptional signatures and related protein levels of splenic
PMN‐MDSCs in vehicle‐ and ruxolitinib‐treated mice at day 7 after
transplantation. (a) Gene pathways that were differentially expressed
in PMN‐MDSCs from two groups according to Gene Set Enrichment analysis.
Gene sets were considered statistically significant at an FDR P‐value
< 0.05 (n = 2 per group). (b) Dot graph shows the alterations of
enriched GO pathways between two groups. The expression profile of
PMN‐MDSCs function‐related genes between two groups according to (c)
RNA sequencing and (d) real‐time PCR (n = 2 or 3 per group). (e) Flow
cytometric detection of ROS in PMN‐MDSCs of vehicle‐ and
ruxolitinib‐treated hosts on day 7 post‐transplantation. Representative
DCFDA staining flow cytometry data gated on CD11b^+Ly6G^+Ly6C^lo cells
are shown. Geometric mean fluorescence intensity (MFI) values are
plotted (n = 3). (f, g) The expression of pSTAT3 and pSTAT5 in
PMN‐MDSCs of vehicle‐ and ruxolitinib‐treated hosts on day 7
post‐transplantation by phosflow techniques (n = 3). (h) The
phosphorylation levels of p65, ERK, p38 and Akt were quantified by
western blot assay in cell lysates of PMN‐MDSCs from vehicle‐ and
ruxolitinib‐treated mice on day 7 post‐transplantation. β‐Actin was
used as an internal control. Data are expressed as mean ± standard
error (SE). **P < 0.01, ***P < 0.001, ****P < 0.0001. These results are
representative of three independent experiments.
Enhanced ROS generation in PMN‐MDSCs isolated from ruxolitinib‐treated mice
via the upregulation of the NF‐κB/MAPK‐p38 signalling pathway in vivo
Previous studies have reported that NADPH oxidase (NOX2) is a key
mediator of PMN‐MDSC‐mediated T cell suppression by regulating ROS
activity.[101] ^24 , [102]^25 Therefore, we hypothesised that the
immunosuppressive function of ruxolitinib is mediated by ROS
generation. To test this hypothesis, PMN‐MDSCs were isolated from mice
in the ruxolitinib and vehicle groups at day 7 after transplantation.
ROS levels were determined using flow cytometric analysis of DCFDA
staining, which revealed that the ROS levels in PMN‐MDSC subsets from
the ruxolitinib group were significantly higher than those from the
vehicle group (Figure [103]4e). Considering that ruxolitinib is a
JAK1/2 inhibitor, we also measured the phosphorylation of STAT3, STAT5
and the downstream mediators of the JAK/STAT signalling pathway using
phosflow technology. As expected, ruxolitinib treatment significantly
reduced the phosphorylation levels of STAT3/5 in PMN‐MDSCs
(Figure [104]4f and g). However, the JAK/STAT signalling pathway
contributes to MDSC accumulation and facilitates ROS release in
PMN‐MDSCs.[105] ^26 Accordingly, ruxolitinib treatment may exert a
contradictory effect on PMN‐MDSCs by increasing ROS generation and
inhibiting the JAK/STAT signalling pathway. We then focussed on the
cytokine storm during aGVHD pathogenesis by STAT3 activation along with
simulation of the NF‐κB pathway,[106] ^27 which is also involved in the
expansion and accumulation of MDSCs. Interestingly, ruxolitinib
treatment increased the phosphorylation levels of p65 and MAPK‐p38 but
had no effect on p‐Erk and p‐Akt levels, as determined using western
blot (Figure [107]4h). These results suggested that anti‐inflammatory
effects of ruxolitinib on PMN‐MDSCs may enhance ROS generation through
bypass activation of NF‐κB/MAPK‐p38 pathway during the development of
aGVHD.
Ruxolitinib enhanced the immunosuppression of MDSCs by increasing ROS
production in vitro
Myeloid‐derived suppressor cells were generated by treating BM cells
isolated from C57BL/6 mice with GM‐CSF and IL‐6 for 4 days in vitro,
according to previously described methods. Following treatment, 89.5%
of cells were identified as CD11b^+Gr‐1^+ cells. CD11b^+ cells can be
subdivided into PMN‐ and M‐MDSCs according to cell surface marker
expression and morphological features (Figure [108]5a). To determine
whether ruxolitinib had a direct effect of maintaining the suppressive
function of MDSCs on T cells, the proliferation of T cells stimulated
with CD3/28 beads was analysed in the presence or absence of MDSCs
pretreated with varying concentrations of ruxolitinib (0.1, 1 or
10 μm). As shown in Figure [109]5c, MDSCs pretreated with 10 μm
ruxolitinib exerted stronger suppressive effects on T cells than those
without ruxolitinib pretreatment. Next, we separated the two subsets of
MDSCs from BM‐derived MDSCs pretreated with 10 μm ruxolitinib for 2 h
and found that PMN‐MDSCs pretreated with ruxolitinib had substantially
greater capacity to inhibit T cell proliferation than M‐MDSCs
(Figure [110]5d and e).
Figure 5.
Figure 5
[111]Open in a new tab
Ruxolitinib‐pretreated PMN‐MDSCs displayed remarkable immunosuppressive
function by upregulation of Nox2 to regulate ROS generation via bypass
activating NF‐κB/MAPK‐p38 pathways in vitro. MDSCs were generated in
vitro from BM cells of C57BL/6 mice in the presence of 40 ng mL^−1
GM‐CSF and IL‐6. After 4 days, cells were stained for CD11b and Gr‐1
expression or the distribution of MDSC subsets was gated on CD11b^+
cells by the expression of Ly6C and Ly6G. (a) Data show one
representative flow cytometric analysis and morphology of MDSCs
subsets. (b) In vitro purified PMN‐MDSCs were pretreated with or
without ruxolitinib (0.1 μm, 1 μm, 10 μm) for 2 h and then incubated
with LPS (1 μg mL^−1) in the presence or absence of ROS inhibitor NAC
(1 mm) for another (h). The production of ROS was monitored by DCFDA
flow cytometry in each group (n = 3). (c–e) CFSE‐labelled CD3^+ T cells
(1 × 10^5 per well) were stimulated by CD3/28 beads, then in vitro
induced MDSCs, PMN‐MDSCs and M‐MDSCs were added at different ratios
with or without ruxolitinib pretreated cocultured for 72 h.
Proliferation of CFSE‐labelled CD3+ T cells was measured with flow
cytometry. In vitro purified PMN‐MDSCs were pretreated with or without
ruxolitinib (0.1 μm, 1 μm,10 μm) for 2 h and then stimulated with LPS
(1 μg mL^−1) (n = 3). (f) The immunosuppressive molecules of PMN‐MDSCs
were detected by real‐time PCR (n = 3). The expression of pSTAT3,
p‐p65, p‐p38, p‐ERK and p‐Akt were examined by phosflow analysis (g)
and western blot assay (h). Data are expressed as mean ± standard error
(SE). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. These
results are representative of three independent experiments.
To determine the pharmacological effects of ruxolitinib on ROS
generation, BM‐derived MDSCs were cocultured with or without
ruxolitinib for 2 h followed by incubation with 1 μg mL^−1 LPS for
30 min. Cellular ROS were detected and labelled in the
CD11b^+Ly6G^+Ly6C^lo PMN‐MDSCs by flow cytometry. As shown in
Figure [112]5b, ROS levels were significantly higher in PMN‐MDSCs
pretreated with ruxolitinib. We next pretreated PMN‐MDSCs with NAC, a
ROS inhibitor, which can specifically block ROS production.
Immunosuppressive molecules, such as Nos2, Il‐10, Ho‐1, Nox2, Cox2,
Tgf‐β and Ido, were strongly upregulated in ruxolitinib‐pretreated
PMN‐MDSCs (Figure [113]5f). We also examined variations in related
signalling pathways. Consistent with the results of our in vivo
experiments, ruxolitinib‐pretreated PMN‐MDSCs exerted inhibition of
STAT3 along with activation of NF‐κB/MAPK‐p38 by western blot detection
(Figure [114]5g and h).
Ruxolitinib inhibited the differentiation of MDSCs into mature myeloid cells
in vitro by inhibiting STAT5
After demonstrating the accumulation of MDSCs in the spleens of
ruxolitinib‐treated aGVHD mice, we next determined the effect of
ruxolitinib on the differentiation of MDSCs into mature myeloid cells,
such as dendritic cells and macrophages. BM‐derived MDSCs were
incubated with 10 ng mL^−1 GM‐CSF for 5 days with or without
ruxolitinib. Ruxolitinib‐treated MDSCs showed impaired differentiation
capacity into CD11c^+ and F4/80^+ cells compared with DMSO‐treated
MDSCs (Figure [115]6a and b). The expression of CD80^+, a marker of the
mature phenotype, was markedly decreased in ruxolitinib‐treated MDSCs
compared with that in MDSCs treated with DMSO as a control
(Figure [116]6c). Previous studies have suggested that GM‐CSF activates
the JAK2/STAT5 pathway, thereby decreasing IRF8 transcription and
altering the differentiation and function of MDSCs.[117] ^28 Our
findings indicated that ruxolitinib suppressed the phosphorylation of
STAT5 and increased the expression of Irf‐8 and Bcl‐xl, thereby
inhibiting the differentiation and promoting survival of MDSCs under
GM‐CSF stimulation (Figure [118]6d and e).
Figure 6.
Figure 6
[119]Open in a new tab
Ruxolitinib decreased the differentiation of MDSCs to mature cells via
STAT5 inhibition in vitro. BM‐derived MDSCs in vitro were cultured with
GM‐CSF with or without different concentrations of ruxolitinib (0.1 μm,
1 μm, 10 μm) for 5 days. (a) Representative plots of macrophages
(CD11b^+F4/80^+) and dendritic cells (CD11b^+CD11c^+) were shown. The
percentages of differentiated mature cells (b) and the expression of
costimulatory molecules CD80^+ gated on CD11b^+ cells (c) were
indicated (n = 3). (d) The phosphorylation of STAT5 was examined by
phosflow techniques (n = 3). (e) The levels of transcriptional factors
related to MDSCs differentiation were monitored by real‐time PCR
(n = 3). Data are expressed as mean ± standard error (SE).
***P < 0.001, ****P < 0.0001. These results are representative of three
independent experiments.
Discussion
Myeloid‐derived suppressor cells are predominantly defined by their
capacity to inhibit T cell responses and promote Treg expansion,
thereby exerting protective effects against the development of
aGVHD.[120] ^29 Myeloid cells secrete molecules that stimulate a range
of signalling pathways, including the JAK/STAT pathway which
contributes to the expansion and function of myeloid cells. The results
of the present study demonstrate that ruxolitinib, a JAK1/2 inhibitor,
efficiently ameliorates the symptoms of aGVHD symptoms, thereby
indicating the potential utility of ruxolitinib in the treatment of
patients after allo‐HSCT. Alterations in the number and proportions of
MDSCs may predict treatment responses to ruxolitinib in patients with
aGVHD when assessed in combination with clinical symptoms. We further
evaluated the effect ruxolitinib on the two major subsets of MDSCs in a
mouse model of aGVHD and found that ruxolitinib increases the
immunosuppressive effects of PMN‐MDSCs by increasing ROS generation via
activation of the NF‐κB/MAPK‐p38 pathway in vivo and in vitro. In
addition, inhibition of STAT5 hampered the differentiation of MDSCs.
Further, MDSC depletion reduced the efficacy of ruxolitinib in treating
aGVHD, which indicates that MDSC may play a pivotal role in mediating
the therapeutic effects of ruxolitinib in aGVHD.
Ruxolitinib has been shown to be effective in treating SR‐aGVHD.[121]
^7 In our cohort, patients with SR‐aGVHD who received ruxolitinib
treatment had favorable ORR and higher OS. Recent studies have reported
that ruxolitinib alleviates aGVHD by inhibiting the JAK/STAT signalling
pathway through multiple mechanisms, including inhibiting the
production of proinflammatory cytokines; impairing the differentiation
and maturation of dendritic cells; suppressing alloreactive T cell
activation and promoting Treg expansion; and inhibiting IFN‐γ‐induced
STAT1 phosphorylation in MSCs to maintain immune tolerance.[122] ^6 ,
[123]^30 , [124]^31 However, patients receiving ruxolitinib had a
modestly higher incidence of infections and CMV reactivation.[125] ^7
Further, there are no standardised methods for monitoring treatment
responses to ruxolitinib. Hence, the activation of JAK/STAT signalling
in other immune cell types may account for the anti‐inflammatory
effects of ruxolitinib in aGVHD or other autoimmune diseases[126] ^32
and therefore warrants further investigation.
Myeloid‐derived suppressor cells have been described as a biomarker of
inflammation with remarkable immunosuppressive capacity. The
proportions of MDSCs in the graft and PB after allo‐HSCT may be
associated with the severity of aGVHD and disease‐free survival.[127]
^33 To determine the importance of measuring the proportion of MDSCs in
monitoring treatment responses to ruxolitinib, we measured the
frequencies of primary immunoregulatory PBMCs before and 7 and 14 days
after ruxolitinib treatment. Interestingly, the levels of MDSCs,
particularly PMN‐MDSCs, continued to increase until day 14 in
nonresponders but returned to pretreatment levels by day 14 in
responders. In particular, the frequencies of MDSCs and PMN‐MDSCs were
substantially lower at day 14 in responders compared with those in
nonresponders. However, we did not observe significant alterations in
Treg and M‐MDSC populations, indicating that MDSCs were more sensitive
with the extension of treatment time during aGVHD. We further observed
that the proportions of MDSCs, particularly PMN‐MDSCs, before treatment
were closely associated with treatment responses to ruxolitinib,
indicating that alterations in MDSC levels may help improve the
monitoring of treatment responses to ruxolitinib. Monitoring the
proportion of MDSCs before and during the administration of ruxolitinib
may aid in determining the optimal treatment regimens for specific
patients with aGVHD. Despite these important findings, our study also
has limitations, including the relatively small sample size of patients
treated with ruxolitinib. Further, the retrospective nature of the
current study and data obtained from a single centre may have
introduced patient selection bias into the study analysis. Therefore,
large cohort prospective investigations are warranted to identify
specific subsets of MDSCs as biomarkers with prognostic impact. It is
required further study for a comprehensive evaluation of MDSCs analysis
by several methods, including FACS, immunohistochemistry and
genomic/transcriptomic/methylation profiling and to confirm the pivotal
status of MDSCs on treatment decision‐making for aGVHD.
To determine the effects of ruxolitinib on MDSCs, we evaluated
survival, body weight and clinical scores in an MHC‐mismatched mouse
model of aGVHD. Ruxolitinib administration in vivo increased the
survival of aGVHD mice and had a substantial inhibitory effect on CD4^+
T cells by decreasing the levels of proinflammatory cytokines and
promoting the expansion of Tregs, consistent with previous
research.[128] ^34 We next evaluated the effect of ruxolitinib on the
proportion of MDSCs in vivo. We observed that ruxolitinib increased the
absolute numbers of MDSCs, particularly PMN‐MDSCs, and enhanced the
suppressive effects of PMN‐MDSCs on T cells.
We then focussed on PMN‐MDSCs, an important cellular target through
which ruxolitinib exerts protective effects against aGVHD. However,
there is a lack of studies on the signalling pathways involved in
PMN‐MDSC function. Using transcriptional profiling of PMN‐MDSCs
isolated from ruxolitinib‐treated mice with aGVHD, GO and GSEA revealed
enrichment of genes associated with MAPK signalling, NF‐κB signalling
and ROS activity in PMN‐MDSCs isolated from ruxolitinib‐treated mice.
We also observed significant upregulation of Nox2, which is known to be
a critical regulator of ROS generation. These data demonstrate that
PMN‐MDSCs generate high levels of cytosolic ROS under the activity of
Nox2 to maintain the undifferentiated state with immunosuppressive
properties of MDSCs in the TME as described previously.[129] ^35 ,
[130]^36 However, the mechanisms underlying the regulation of ROS in
PMN‐MDSCs are yet to be fully elucidated. Recent studies have indicated
that the activation of the JAK/STAT pathway leads to the accumulation
of MDSCs and prevents their differentiation into mature cell types. The
activation of STAT3 is directly responsible for upregulating the
transcription of Nox2 and increasing ROS production by MDSCs in
tumor‐bearing mice.[131] ^24 Patients with oral squamous cell carcinoma
have higher proportions of PMN‐MDSCs, with higher levels of ROS and
pSTAT3 signalling abrogating T cell proliferation.[132] ^37
In the present study, ruxolitinib effectively inhibited the activation
of JAK/STAT pathway and promoted the production of ROS in PMN‐MDSCs,
which appears to contradict the findings of previous studies. As aGVHD
is known to trigger the cytokine storm, aGVHD may involve the
activation of several other inflammatory pathways in addition to the
JAK/STAT pathway. Accordingly, other pathways may be involved in
mediating the effect of ruxolitinib on MDSCs. Previous studies have
revealed that ROS production in MDSCs is mediated by activation of the
NF‐κB and MAPK‐p38 signalling pathways in lupus nephritis.[133] ^38
NF‐κB/MAPK‐p38 signalling may also play a critical role in the
accumulation of MDSCs and mediating their activation in response to
TNF‐α, IL‐1β and other inflammatory cytokines.[134] ^39 , [135]^40 In
the context of allo‐HSCT, the myeloid differentiation primary response
gene 88 (MyD88)/NF‐κB pathway may also contribute to the expansion of
donor MDSCs and initiation of aGVHD.[136] ^41 Myd88^−/− MDSCs lose the
ability to suppress T cell activity and release anti‐inflammatory
cytokines such as IL‐10 and Arg‐1.[137] ^42 In the present study, we
confirmed that upregulation of the NF‐κB/MAPK‐p38 pathway is involved
in maintaining MDSC function in ruxolitinib‐treated PMN‐MDSCs in vivo
and in vitro, which is consistent with the results of our transcriptome
analysis.
The JAK/STAT signalling pathway may play a critical role in maintaining
MDSC numbers and blocking their differentiation into mature cells.
GM‐CSF triggers Tyr phosphorylation of the GM‐CSF receptor resulting in
JAK/STAT5 activation and downregulation of Irf8, thereby promoting MDSC
differentiation and survival.[138] ^43 Our results indicate that the
inhibition of pSTAT5 by ruxolitinib may block the differentiation of
immature MDSCs into macrophages or dendritic cells via upregulation of
Irf8 and prevent apoptosis in MDSCs by upregulating Bcl‐2.
In conclusion, we evaluated the therapeutic effect of JAK1/2 inhibitor
ruxolitinib in patients with SR‐aGVHD after allo‐HSCT and demonstrated
that the monitoring of MDSCs is useful in assessing treatment responses
to ruxolitinib and may aid in selecting patients suitable for
alterations in treatment regimen. Using a mouse model of aGVHD and in
vitro experiments, we observed that two signal transduction pathways
mediate the expansion of MDSCs in response to ruxolitinib therapy. On
the one hand, induced by proinflammatory cytokines, ruxolitinib could
promote the differentiation of MDSCs into PMN‐MDSCs, which displayed
strong immunosuppressive function by upregulating Nox2, thereby
regulating ROS generation through bypass activation of NF‐κB/MAPK‐p38
pathways. On the other hand, MDSCs also required second signals to
maintain their undifferentiated state via STAT5 influenced by
ruxolitinib. The results of the present study demonstrate the direct
immunoregulatory effect of ruxolitinib on MDSCs may also be relevant to
pathological conditions other than aGVHD and facilitate the development
of novel strategies for modifying MDSCs in the treatment of aGVHD.
Patients exposed to long‐term treatment with ruxolitinib should be
closely monitored for infectious complications or relapse of the
underlying disease. However, considering their potential as therapeutic
targets, the JAK/STAT and other signalling pathways involved in MDSC
functional polarisation and phenotype warrant further investigation.
Further studies are required to define the interactions between
effector immune cells and MDSCs, which may improve therapeutic
responses and disease outcomes.
Methods
Patient enrolment and data collection
We retrospectively analysed data from 74 patients with SR‐aGVHD who
received ruxolitinib as second‐line therapy following allo‐HSCT at the
Institute of Haematology, Chinese Academy of Medical Sciences, China,
between May 2017 and December 2020. Eligibility criteria were aGVHD
progression after 3 days or incomplete response after 7 days (SR‐aGVHD)
of treatment with methylprednisolone at a dose of 2 mg/kg per day.[139]
^44 aGVHD grading was evaluated according to MAGIC criteria.[140] ^45
The clinical characteristics of patients are shown in Supplementary
table [141]1. All patients were followed up through telephone and
outpatient appointments until 31 January 2022.
The initial dose of ruxolitinib was 5 mg twice daily, and in cases with
good tolerance and stable haematological parameters, the dose was
increased to 10 mg twice daily at the physician's discretion. Based on
a previous study,[142] ^7 the safety and efficacy of ruxolitinib was
evaluated at 28 days after treatment initiation with ruxolitinib.
Treatment responses were classified as complete response (CR), partial
response (PR) or treatment failure.[143] ^46 The skin, GI and liver
were the main target organs involved in aGVHD. CR was defined as the
resolution of all clinical symptoms of GVHD. PR was defined as an
improvement in at least one organ or site without progression at any
other organ or site. Treatment failure was defined as the absence of
improvement in aGVHD, disease progression in any organ or the need to
start a new treatment for disease control. Overall survival (OS) was
measured from the initiation of ruxolitinib treatment until death from
any cause. Adverse events in patients who received ruxolitinib are
summarised in Supplementary table [144]2. The severity of cytopenia was
defined according to previously published criteria.[145] ^47
Peripheral blood (PB) samples were collected from patients with
SR‐aGVHD who received ruxolitinib treatment between June and October
2020. Seven patients achieved PR or CR (responders), and six showed
treatment failure (nonresponders). MDSCs and Tregs were isolated from
PB mononuclear cells (PBMCs) using Ficoll–Hypaque density gradient
centrifugation on the day before treatment initiation with ruxolitinib
and at 7 and 14 days after treatment initiation. This study was
approved by our centre's Medical Ethics Committee (Ethical approval No.
IIT2020016‐EC‐3) and conducted in accordance with the Helsinki
declaration. Written informed consent for collection data was obtained
from each patient or their legal representative prior to enrolment.
Mice
C57BL/6 and BALB/c mice aged 8–9 weeks were purchased from Huafukang
Company (Beijing, China) and maintained in specific pathogen‐free
barrier facilities. All experiments were performed in accordance with
protocols approved by the Institutional Animal Care and Use Committee
of the Institute of Hematology, Chinese Academy of Medical Sciences.
Mouse models of bone marrow transplantation and aGVHD
Recipient BALB/c mice received total body (^60Co source) irradiation at
a dose of 8 Gy, which was split into two doses and applied at an
interval of 4 h on day −1. Irradiated recipient mice were then
intravenously injected with 1 × 10^7 bone marrow (BM) nucleated cells
with or without 3 × 10^7 spleen cells obtained from age‐ and
gender‐matched C57BL/6 mice within 24 h on day 0. BM and splenocyte
suspensions were obtained from iliac, femoral, tibial and splenic
samples. aGVHD scores were calculated according to the weight, posture,
activity, fur texture and skin appearance of mice, as described
previously.[146] ^48 Liver, intestine and colon specimens were
collected on day 7 after transplantation. Specimens were fixed with 4%
neutral formaldehyde, embedded in paraffin, sectioned, stained with
haematoxylin and eosin and observed under a light microscope. In some
experiments, mice were intraperitoneally injected with 200 μg of
anti‐Gr‐1 depleting antibodies (BioXcell, New Hampshire, USA) every
other day between days 5 and 29.
Treatment with ruxolitinib
The mouse models of aGVHD were administered with ruxolitinib (Novartis,
Basel, Switzerland) dissolved in PEG300 and 5% dextrose (at a ratio of
1:3) by oral gavage at a dose of 30 mg kg^– ^1 twice daily from day 0
until day 30, as described previously.[147] ^6 The vehicle group was
only administered with PEG300:5% dextrose (1:3). For the in vitro
study, MDSCs were pretreated with various concentrations of ruxolitinib
(0.1, 1 and 10 μm), which was diluted with dimethyl sulfoxide (DMSO)
for 2 h.
Isolation and generation of MDSCs
Total MDSCs (CD11b^+Gr‐1^+), PMN‐MDSCs (CD11b^+Ly6G^+Ly6C^lo) and
M‐MDSCs (CD11b^+Ly6G‐Ly6C^hi) were isolated from single‐cell
suspensions of splenic samples using a FACS Arial III cell sorter. The
purity was typically over 90%. For in vitro experiments, MDSCs were
generated from BM cells obtained from C57BL/6 wild‐type mice and
maintained in culture medium supplemented with 40 ng mL^−1 murine IL‐6
and 40 ng mL^−1 GM‐CSF (Peprotech, Rochy Hill, USA) for 4 days.[148]
^49 The purity ranged between 80% and 90%, as assessed via flow
cytometry. PMN‐ and M‐MDSC subpopulations were purified using MDSC
Isolation Kit (Miltenyi Biotec, Kӧln, Germany) or FACS Arial III cell
sorter.
Myeloid‐derived suppressor cell suppression assay
Splenic CD3^+ T cells isolated from C57BL/6 mice using CD3 microbead
kit (Miltenyi Biotec) were incubated with 5 μm CFSE (Biolegend, San
Diego, USA) for 8 min in phosphate‐buffered saline (PBS) at room
temperature and then washed with RPMI 1640 containing 10% foetal bovine
serum (FBS). Next, 1 x 10^5 labelled T cells were seeded into 96‐well
round‐bottom plates with anti‐CD3/CD28 beads (Invitrogen, New York,
USA) and cocultured with or without different ratios of purified MDSCs,
PMN‐MDSCs or M‐MDSCs in RPMI 1640 supplemented with 10% FBS, 1%
penicillin/streptomycin and 50 ng mL^−1 recombinant IL‐2 for 72 h. Cell
proliferation was measured using flow cytometry. Cell suppression (%)
was calculated using the following formula:
[MATH:
%CSFE−diluted
cells without
MDSCs−%CSFE−di
luted cells with
MDSCs%CSFE−diluted cells without
MDSCs−%CSFE−di
luted cells without
stimulators×100%
:MATH]
Myeloid‐derived suppressor cell differentiation assay
BM‐derived MDSCs were cultured in the presence of 10 ng mL^−1
recombinant GM‐CSF (Peprotech) for 5 days. In some experiments, cell
cultures were treated with varying concentrations of ruxolitinib (0.1,
1, or 10 μm) or DMSO. Subsequently, cell phenotypes were assessed using
flow cytometry.
Flow cytometry
For the analysis of cell surface molecules, human blood cells were
isolated and fluorescently stained for 30 min at 4°C in the dark with
the following antibodies: APC‐Cy7 anti‐human CD4, PE‐Cy7 anti‐human
CD8, APC anti‐human CD25, PE anti‐human CD11b, FITC anti‐human HLA‐DR,
PE/Cy7 anti‐human CD15 and APC/Cy7 anti‐human CD14 (Biolegend). In
mice, single‐cell suspensions were prepared from BM, spleen and cell
cultures in vitro. The following monoclonal antibodies were added to
the cell suspensions: Percp/Cy 5.5 anti‐mouse H2‐Kb, APC anti‐mouse
CD11b, PE anti‐mouse Gr‐1, APC‐Cy7 anti‐mouse ly6C, PE‐Cy7 anti‐mouse
Ly‐6G, PE anti‐mouse CD69, APC anti‐mouse CD25, APC‐Cy7 anti‐mouse CD4,
PE‐Cy7 anti‐mouse CD8, Percp/Cy5.5 anti‐mouse CD11b, APC anti‐mouse
CD80, PE‐Cy7 anti‐mouse CD11c and PE anti‐mouse F4/80 (Biolegend). For
Foxp3 staining, cells were resuspended in fixation/permeabilisation
buffer (eBioscience, New York, USA) according to the manufacturer's
protocol. Cells were incubated with Foxp3 antibodies at a concentration
of 1:100 at room temperature for 1 h under protection from light. For
intracellular cytokine staining, cells were stimulated with cell
stimulation cocktail (500x, Invitrogen) for 4–5 h and blocked with
Brefeldin A solution (1000×, Invitrogen). For phosphorylate staining,
cells were fixed with IC fixation buffer (Invitrogen) and then exposed
to precooled methanol before incubation with pSTAT3 and pSTAT5
antibodies. Viability was assessed using Fixable Viability Dye EF506
(Invitrogen). Finally, stained cells were washed with PBS containing 2%
FBS or permeabilisation buffer (eBioscience) and analysed using FACS
Canto II. Data were analysed using FlowJo software.
Reactive oxygen species measurements
Myeloid‐derived suppressor cells were incubated in serum‐free medium at
37°C in the presence or absence of 1 μg mL^−1 LPS (Sigma, St. Louis,
USA) for 30 min. Intracellular ROS levels were measured via flow
cytometry using 2.5 mm DCFDA, an oxidation‐sensitive dye (Beyotime
Biotechnology, Shanghai, China). For determining the inhibition of ROS,
cells were incubated with 1 mm N‐acetyl‐L‐cysteine (NAC; Beyotime
Biotechnology) for 2 h.
Reverse transcription real‐time polymerase chain reaction (RT–PCR) and bulk
RNA sequencing (RNA‐seq)
Total RNA was extracted using TRIzol reagent (Invitrogen) according to
the manufacturer's instructions. Reverse transcription was performed
using One‐Step RT–PCR SuperMix (Transgen. Inc, Beijing, China) to
synthesise cDNA. Quantitative RT–PCR was performed using GeneAmp 7500
Sequence Detection System (Applied Biosystems). The relative abundance
of each gene was calculated using 2^−ΔΔCT method upon normalisation to
β‐actin expression. RNA‐seq was performed using the BGIseq500 platform
(BGI, Wuhan, China). The PCR primers used in this experiment are listed
in Supplementary table [149]3.
Western blot
Cells were lysed with RIPA lysis buffer (Beyotime Biotechnology)
containing protease and phosphatase inhibitors according to standard
techniques.[150] ^50 Antibodies against phospho‐p38 MAPK, phospho‐Akt
(Ser 473), phospho‐p44/42 MAPK (Erk1/2 and Thr202/Tyr204),
phospho‐NF‐κb p65 (Ser 536) and HRP‐linked anti‐rabbit IgG antibodies
were purchased from Cell Signaling Technology (Danvers, USA). Protein
bands were visualised using ECL plus chemiluminescent substrate
(Invitrogen). β‐actin was used as the internal control.
Cytokine profiling
Serum was collected from at least three mice per group. Supernatants
were collected after centrifugation of the culture media from MDSCs
from control and ruxolitinib‐treated mice cocultured with T cells.
According to the manufacturer's instructions, cytokine profiling was
performed using the LEGENDplex™ Mouse Inflammation Panel (Biolegend).
Measured levels of mouse cytokines are reported in ng mL^−1.
Statistical analyses
Statistical analyses were performed using GraphPad Prism 9.0 and R
4.0.0. Results are reported as the mean ± standard error of the mean.
The Student's t‐test was used to compare differences between two
groups. One‐ or two‐way ANOVA was used to compare multiple groups. The
correlation between categorical variables was calculated using
Spearman's correlation. Survival curves were estimated using the
Kaplan–Meier method and compared using the log‐rank test. A P‐value of
< 0.05 was considered statistically significant: *P < 0.05, **P < 0.01,
***P < 0.001 and ****P < 0.0001.
AUTHOR CONTRIBUTIONS
Yigeng Cao: Conceptualization; data curation; formal analysis; funding
acquisition; writing – review and editing. Jiali Wang: Data curation;
formal analysis; methodology; writing – original draft. Shan Jiang:
Data curation; formal analysis. Mengnan Lyu: Formal analysis;
methodology. Fei Zhao: Data curation; formal analysis. Jia Liu: Data
curation; formal analysis. Mingyang Wang: Investigation; methodology.
Xiaolei Pei: Methodology; resources; software. Weihua Zhai: Data
curation; funding acquisition. Xiaoming Feng: Resources; validation.
Sizhou Feng: Data curation; investigation. Mingzhe Han: Methodology;
supervision; visualization. Yuanfu Xu: Funding acquisition;
methodology; supervision; writing – review and editing. Erlie Jiang:
Conceptualization; funding acquisition; investigation; project
administration; writing – review and editing.
Conflict of interest
The authors declare no competing interests.
Supporting information
Supporting information
[151]Click here for additional data file.^ (5.8MB, docx)
Acknowledgments