Discussion:
Allogeneic hematopoietic stem cell transplantation is an effective
treatment for patients with hematologic malignancies who have
HLA-compatible or incompatible donors(25). Patients are prepared with
chemotherapy or radiotherapy and then hematopoietic stem cells are
injected intravenously. Shortly after injection, hematopoietic stem
cells leave the bloodstream and are replaced in the extravascular space,
thereby cells are transplanted into the patient’s bone marrow. During a
successful transplant, hematopoietic stem cells proliferate and begin
hematopoiesis in the patient’s bone marrow. Hematopoietic stem cell
transplantation depends on several factors, including: the severity of
the preparation regimen, the dose of the transplanted stem cells, the
degree of incompatibility, the T-cell graft content, and the severity of
post-transplant immune suppression(26, 27) . However, a major problem in
HSCT patients is graft-versus-host disease that occurs in both acute and
chronic forms and can be the leading cause of death in these
patients(28). Grading systems have been developed to determine the
severity of GvHD; patients with Grade III and IV aGvHD have been
estimated to have a 5-year survival of 25% and 5%, respectively, and
an increase in cGvHD severity is correlated with increased mortality
(1). Other major causes of mortality are relapse (approximately 30% of
deaths) and infection (approximately 10% of deaths), and most deaths
occur within the first two years after transplantation (28, 29) .
Various therapeutic strategies have been discovered to reduce the
incidence of GvHD and recurrence, one of them is the use of MSCs as a
cell-based approach. Mesenchymal stem cells are multipotent progenitors
that can be isolated from various sources such as adult bone marrow,
adipose tissue, and various embryonic tissues and have the ability to
differentiate into different cell and tissue lineages such as
chondrocytes, osteoblasts, and adipocytes. However, cord blood and bone
marrow are the most common sources of MSCs for clinical use. MSCs have
been shown to release numerous colony-stimulating factors and cytokines,
both permanently and after stimulation. These cells are also able to
support the prolonged proliferation of initiating culture cells in vitro
and to increase the engraftment in preclinical models (30, 31). In
addition, MSCs have been shown to have potent suppressive activity that
actually targets all types of blood cells(32-34). Since proliferative
MSCs in the culture medium can be injected intravenously without
toxicity, these pre-clinical studies have rapidly developed to
therapeutic applications that increase HSCT engraftment and reduce the
incidence and severity of GvHD(35, 36). In the extracellular
environment, MSCs enhance hematopoiesis and inhibit Tcell proliferation,
NK cell cytotoxicity, and dendritic cell differentiation(25).Previous
studies have shown that co-injection of MSCs with HSCs can support and
improve transplantation outcomes and prevent severe acute GVHD.
Pre-clinical studies have shown improved engraftment of HSCs and reduced
risk of GvHD after simultaneous injection of proliferated MSCs and
HSCs(27, 37-39) and this has led to further clinical studies and
investigations based on MSC therapy with the aim of improving the
outcome of allogeneic HSCT transplantation in patients with hematologic
malignancies. However, although numerous clinical and preclinical
studies have been performed with MSCs, the efficacy of MSCs in
transplantation conditions is still unclear. The aim of this study was
to evaluate the effect of co-injection of MSCs with HSCs in allogeneic
transplantation of patients with hematologic malignancies for further
analysis of GvHD and engraftment. The results of this study showed that
there was no significant difference in WBC and platelet engraftment
between two groups of study, however, faster engraftment was seen in
group of MSC+HSC transplantation. MSCs produce cytokines that support
hematopoiesis and thus can potentially enhance bone marrow recovery such
as G-CSF that can reduce the effects of MSC injection on
transplantation(26). In a meta-analysis study conducted by Merete
Kallekleiv et al. In 2016, the findings of several clinical trials were
evaluated and summarized to assess the effect of co-injection of MSC and
HSCs on transplant outcomes such as engraftment, GVHD, relapse. And the
results showed that when MSC was injected within 24 post-HSCT, it did
not improve neutrophil engraftment time and no significant statistics
was observed (26). Kaiyan Liu and colleagues showed in a study that
co-injection of MSCs and HSCs resulted in a faster increase in platelet
concentration (500 × 109 cells / L)(29). In this
study, as in the present study, there was no significant difference
between platelet engraftment in the treatment and control groups.
However, over a period of 100 days, the time to reach platelet count to
50 × 109 cells / L in the treatment group was
significantly shorter than the control group. In a study by Ball et al.
(2008), recovery of lymphocytes, especially NK cells, was faster in
patients receiving MSCs than in control patients(35). In the present
study, there was no significant difference in WBC engraftment between
two groups. In this study, it was hypothesized that maybe the
accelerated WBC engraftment time be dwindled by administration of G-CSF
in the early post-transplant phase. As Ringden et al.(40) have observed,
treatment with G-CSF accelerates neutrophil but not platelet
engraftment. In another study conducted by Wu et al. (2013), cord blood
derived MSCs were shown to accelerate neutrophil and platelet
engraftment in recipient of HSCs with the same source. This study has
shown that patients receiving UCMSCs not only have faster engraftment
compared to cord blood HSC alone, but also the clinical application of
third donor proliferated UCMSCs is safe and feasible. This study
suggests that MSCs play an important role in stabilizing specific bone
marrow-specific microenvironment for hematopoiesis by providing
appropriate scaffolding and secretion of different cytokines, adhesion
molecules, and extracellular matrix proteins and it be mentioned that
chemotherapy and radiotherapy pre-transplantation can damage bone marrow
stroma(34, 41). Simultaneous injection of MSC and HSC can have a major
impact on bone marrow regeneration, especially when the number of
available HSCs is limited like cord blood HSC transplantation. As
Macmillan reported in a 2009, transplantation of proliferated BM-MSCs
originated from haploidentical parents lead to better engraftment in
pediatric transplantation with cord blood HSCs(42). In a study by
Frassoni et al and colleagues, they simultaneously injected mesenchymal
stem cells derived from patients (identical donors for HLA) and
hematopoietic stem cells to enhance HSC engraftment. Their results
showed a higher platelet count at day 50 after transplantation in the
treated group with MSC and HSCs(43). These results may be due to MSCs’
support for hematopoietic microenvironment and/or release of soluble
factors to enhance HSC engraftment. MSCs have been shown to be able to
regenerate bone marrow after direct injection into the BM (43, 44). In
addition, after systemic injection of MSCs in animal models, they have
been identified BM and can enhance the engraftment of HSCs (45-47).
These results suggest that MSCs are able to migrate to bone marrow and
support hematopoiesis. However, other recent studies have suggested that
based on the observations of transient and weak MSCs engraftment after
systemic injection, soluble factors released by MSCs may also play a
role in HSCs engraftment. MSCs have been shown to produce several growth
factors that improve HSC growth and differentiation, as well as the
chemokines and chemokine ligands that are important in migration of HSCs
into bone marrow (48-51). In another study, K Le Blanc and colleagues
treated 7 patients with MSCs with allogeneic HSCT. And reported that
co-infusion of MSCs and HSCs results in rapid neutrophil and platelet
engraftment and 100% chimerism, and subsequently reduces the likelihood
of severe acute GVHD and infections(7). In one study, Le Blanc used
co-injections of MSCs and HSCs for 3 patients with previous failure in
transplantation and 4 patients with SAA in order to increase the
engraftment, but the engraftment was not compared with any control
group(42). The results of a study by Lynne M. Ball et al. (2007) showed
that simultaneous injection of MSCs and HSCs could regulate
alloreactivity in donor and enhance the engraftment of HSCs to the BM of
recipient and thereby reduce the risk of graft failure(52). The results
of these two studies were different from those of the present study,
which may be due to the low number of patients in our study and further
studies are needed to determine the precise impact of MSC on
engraftment. Although there are still differing views on the effects of
MSCs on HSC engraftment, most published data have shown that
simultaneous injection of MSCs and HSCs is feasible and safe (53). As
stated previously, in in vitro condition, MSCs support hematopoiesis and
on the other hand, inhibit T cell proliferation, NK cell cytotoxicity,
and dendritic cell differentiation(27). In animal models, co-injection
of MSCs and HSCs has been shown to induce long-term post-allograft
survival in baboons and also to prevent GvHD in some mouse models as
well as in aGvHD xenograft models (human to NOD / SCID mice) by
increasing the production of regulatory T cells(28, 29, 43). Past
studies have shown that co-administration of stromal cells may support
hematopoietic / lymphopoietic donor transplantation and prevent severe
aGvHD(54). Some studies have shown that injections of MSCs can treat
severe GVHD. Immune system-mediated inhibition of mesenchymal stem cells
is a complex mechanism involving changes in the maturation of
antigen-presenting cells and in the system of cytokine secretion and
inhibition of monocyte differentiation into dendritic cells. These cells
induce immunosuppressive regulatory lymphocytes and CD8 apoptosis and
thus inhibit the immune system(38). In the present study, the symptoms
and grade of GVHD were more severe in the control group than in the
mesenchymal stem cells treated group. But there was no statistically
significant difference in the incidence of GVHD between the two groups.
The reason for the lack of significant difference can be due to the low
sample size. However, the incidence of aGvHD in MSC contained group was
shorter in 5 treated patients compared to the control group. The results
of this study are comparable with previous reports in patients
undergoing allogeneic transplantation of HLA-compatible siblings(46-49,
55).
In a meta-analysis study performed by Hillard M. Lazarus et al.
“Evaluating the overall incidence of acute and chronic GVHD”, acute
GVHD (grade 1-2 or grade 3-4), and chronic GVHD (classified into two
limited and widespread groups), no significan no statistically
significant difference between the two groups in the treated and treated
mesenchymal stem cells. In this study, they believe that co-injection of
cultured MSCs with HSCs is safe and possible. However, evaluation of the
appropriate dose of MSCs and the number of injections to prevent or
treat GVHD during allogeneic HSCt in phase 2 clinical trials should be
performed(27), and also in a study by Kaiyan Liu and colleagues its
suggested that MSCs injection was not effective in preventing GVHD. They
stated several reasons for this finding. As shown in previous studies,
the main source of patients post-transplant MSCs is from host. So, MSCs
can only inhibit the patient’s immune system for a specific period after
injection. Therefore, it is difficult to see the positive effects of
MSCs when the patient’s immune system is severely inhibited. The
mechanism of immune suppression by mesenchymal cells in the body is also
unclear, and further studies are needed to determine the amount of
cytokines produced by GVHD-associated mesenchymal stem cells to
determine whether injections of mesenchymal cells can effectively
prevent the GVHD or not(29). In another study, Y. Tian and his
colleagues reported different results. Their results showed that
co-injection of MSCs and HSCs into the body could act as an immune
suppressor and protect the allogeneic graft receptors against GVHD and
prolong their survival time. They said the T cell homeostasis and
enhancement of CD4 + CD25 + T cell proliferation are the main reasons
for this result. In another study conducted by F. Baron et al on 20
HLA-incompatible patients, it was shown that injection of MSCs 2 to 0.5
h before allogeneic transplantation led to a reduction in the incidence
of aGvHD grade IV and is comparable to its incidence rate in patients
undergoing allogeneic transplantation from HLA-compatible unrelated
donors (without MSCs)(27). The results of this study suggest that MSCs
can prevent GvHD related death in patients receiving PBSCs from
HLA-compatible unrelated donors. In another study by Ning et al in 2008,
they compared the co-injection of MSCs and HSCs in 10 patients with a 15
controls. It was shown that just one patient in MSC receiving group
showed grade II GvHD compared to 8 controls. The overall incidence of
aGvHD in MSC receiving group was 44% and in the control group was 73%.
In this study, it was shown that co-injection of MSCs at even lower
doses than previously reported (0.3-1.5×106 cells/kg)
is able to prevent GvHD(54). These findings indicate that MSCs can have
clinical applications for the engineering of transplantation strategies
in allogeneic HSCT (39). In a study, K Le Blanc and colleagues treated 7
patients with MSCs and HSCs. None of the patients had GVHD. In this
study the reason for the lack of severe GVHD was the effect of MSCs on
immune system regulation and donor cell chimerism(54). According to
several mouse transplantation studies that have shown the role of MSCs
in tumor growth in vivo (56, 57), adverse effects of co-injection of
MSCs and HSCs with immunosuppression have been investigated in other
studies. In fact, MSCs are able to inhibit allogeneic T cell responses
and suppress T-cell proliferation by cell-cell interactions and soluble
factors (58-61). Suppressed T-cell function is able to attenuate or
eliminate graft versus leukemia (GVL) in allograft conditions. There is
no evidence showing that MSCs were able to selectively suppress GvHD but
did not affect GVL(62).
In melanoma, co-injection of MSCs and allogeneic tumor cells results in
faster tumor growth, even when MSCs migrate to distant areas. This
suggests that MSCs may suppress systemic immune function(56). Another
report showed a mismatch between the in vitro and in vivo behavior of
MSCs. MSCs inhibit the proliferation of malignant cells with
hematopoietic and non-hematopoietic origin in vitro, by stopping tumor
cells in the G1 phase of the cell cycle. However, when tumor cells plus
MSCs were injected into NOD / SCID mice, tumor growth was faster than in
the group receiving only tumor cells. The authors propose that MSCs may
maintain the ability of self-renewal of cancer cells and this is a new
mechanism by which the stromal environment can affect the process of
malignant disease(63). The results of this study showed no significant
difference in recurrence after transplantation. In the MSC recipient
group, one patient died of recurrence and one patient died of GvHD,
while in the control group consisted of 5 patients, 2 patients died
because of GvHD and one patient died because of severe immunosuppression
related infection due to immunosuppressive therapy. So the results
showed no significant difference between MSC injection and recurrence.
In a study by Baron et al with 20 patients, co-injections of MSCs and
HSCs did not eliminate the effects of GVT, and a one-year recurrence in
the MSC-treated group was reported 30% and it was similar to the
recurrence in the control group(31). There was no significant difference
between the two groups in the study by Lee et al(64). The only study
that reported a significant difference between the MSC group and the
control group was by Ning et al. with 20 patients in 2008(65). The
clinical trial reported that patients receiving MSC had a significantly
higher rate of relapse (60% vs 20% in the control group). In addition,
the median time from transplantation to recurrence was shorter in MSC
recipient group than in the control group (63 day vs 177 days). In fact,
the study by Ning and his colleagues confirmed some of the previous
findings that MSC may help tumor growth. Plasma biomarkers are important
factors in the diagnosis of acute GVHD after HSCT. Studies have recently
been conducted to find and validate the biomarkers associated with GVHD.
In this study, the levels of TNFR1, ST2 and DNAM-1 markers were measured
on days 7, 14 and 28 post-transplantation for prediction of GVHD.
Several biomarkers (or combinations of biomarkers) have been associated
with greater chance of GvHD occurrence, clinical improvement of GvHD
symptoms, steroid resistance, and overall survival(56, 66, 67). For this
reason, a panel of soluble factors that have previously been reported to
be involved in GvHD was evaluated in this study. DNAM-1 is a member of
the immunoglobulin family and is expressed permanently in most CD4 + T
cells, CD8 + T cells, NK cells, and monocytes(32, 33). DNAM-1 ligands
are CD155 and CD112 that are expressed on hematopoietic and
non-hematopoietic cells including epithelial and endothelial cells(31,
64). Interestingly, the expression of CD155 and CD112 is regulatory
increased with DNA damage response pathways in response to
chemotherapy(65). Interaction between DNAM-1 and CD8 + T cells and NK
cells and its ligand on the surface of target cells increase the
cytotoxicity of cells(32, 66). In addition, DNAM-1 is involved in a
range of T cell functions. Various studies have shown the critical role
of DNAM-1 in the development of aGvHD in mice(18, 24). A study by Kanaya
et al. (2008) showed that serum sDNAM-1 concentration prior to
allogeneic hematopoietic stem cell transplantation was associated with
the development of aGvHD, suggesting that sDNAM-1 is a unique biomarker
for prediction of aGvHD. The study of 71 patients found that the
incidence of GVHD in patients with high levels of soluble DNAM-1 at 7
days before transplantation was significantly higher than the patients
with lower levels of DNAM-1. Their results showed that soluble DNAM-1
can be considered as a predictive marker of acute GVHD(38). In the
present study, the criteria for selecting patients was having the DNAM-1
level more than 7 ng/L. In this study, out of 10 enrolled patients, 5
patients faced aGvHD (3 patients in control group and 2 patients in MSCs
recipient group). Given the limited number of samples in this study, it
was not possible to measure the specificity and sensitivity of this
marker to predict GvHD. In a study carried out by Kanaya et al. it was
shown that DNAM-1 level pre transplant (-7_0 days) for sensivity of
GvHD is 43% and for specificity is 82.6%. In this study, 60% of
control group and 40% of MSC recipient patients showed GvHD of the
first 10 patients who initially had high level of DNAM-1. Given the role
of MSCs in reducing GvHD occurrence, this marker is likely to be more
sensitive in this study than the previous studies. However, only high
DNAM-1 before allogeneic transplantation may not be sufficient to
accurately predict aGvHD, and a combination of other predictive markers,
coupled with high sDNAM-1 prior to allogeneic transplantation, will
enable clinicians to Treatment of allo-HSCT patients with stronger
preventive therapies for aGvHD. Although the results suggest that
sDNAM-1 is produced by proteolytic degradation induced by MMP, the
precise mechanism of sDNAM-1 production remains unclear. In addition,
why healthy individuals show a wide range of serum sDNAM-1 levels and
whether this range is due to the variability of MMP activity between
healthy individuals is unclear. Past studies have shown that MMP
activity is determinant in the development of aGvHD (14). For this
reason, regarding the role of DNAM-1 in GvHD, it is hypothesized that
patients with high MMP activity, which may have high serum levels of
DNAM-1, have a high risk for aGvHD after allo-HSCT. DNAM-1 may also bind
to CD155 on the surface of antigen-presenting cells or tissue of target
organs. Since DNAM-1 has a ligand in common with TIGIT and CD96(68),
this may interfere with the interaction of CD155 with TIGIT and CD96.
Unlike DNAM-1, TIGIT and CD96 generate T cell inhibitory signals and
suppress activation of effector T cells. Consequently, binding of DNAM-1
to CD155 suppresses the inhibitory signals generated by these two
immunoreceptors in T cells, leading to increased T cell activity and
worsened aGvHD. In fact, CD155 deficient mice receiving allogeneic
hematopoietic grafts show increased risk of GvHD(69). Post-transplant
DNAM-1 level did not differ significantly between the two groups on
different days. However, a decrease in the level of DNAM-1 in a study by
Nabekura in 2010 showed that DNAM-1 increases the proliferation of
active alloreactive CD8 + T cells and the production of IFNγ by these
cells(70). Past studies have also shown the important roles of this
marker in modulating cellular immunity such as: 1) Increasing the
cytotoxicity through cytotoxic T lymphocytes (CTLs) (including
alloantigen-specific CTLs) and NK cells against target cells expressing
CD155 and CD112 and increased secretion of cytokines such as IFNγ, 2)
Proliferation and differentiation of CD4 + naive T cells to Th1 cells in
corporation with LFA-1(71-74) . Given the suppressive role of MSCs, it
can be implied that MSCs interfere with APCs and reduce antigen
presentation to T cells, so it interfere the DNAM-1 stimulus function
and consequently reduces the IFNα production by cells and decrease the
inflammatory conditions. The level of TNFR1 at day 7 was higher in the
GVHD group than in the non-GVHD group. In our study, the mean ST2 level
was lower in MSC contained group, as it was decreased after +7 days. ST2
is a soluble receptor for interleukin-33 and is released from activated
T cells during GVHD development and progression. In other words,
elevated ST2 in the patient’s blood indicates progression of GVHD. In a
study by Matthew J. Hartwell and colleagues, an algorithm was designed
based on biomarkers of patients 7 days after transplantation to identify
the patients with high risk of GVHD and NRM (69). The decrease in ST2
levels from day +7 in patients treated with MSCs indicates the positive
effect of these cells on immune suppression and decrease in the severity
of GVHD in these patients. In another study by Mark T. Vander Lugt et
al, ST2 was identified as the most significant marker for
treatment-resistant GVHD and recurrent and death without recurrence. In
this study, patients with high ST2 levels were 2.3 times more likely to
develop refractory GVHD and relapse-free death compared with patients
with low ST2 levels at baseline (75).