Deep thinking: how to view the immune regulation of mesenchymal stem cells

Starter: Mesenchymal stem cells

It is well known that mesenchymal stem cells (MSCs) have a certain different class of
effects on immune cells involved in both innate and adaptive immunity.
However, the author believes that it is still confusing and cannot explain the phenomenon
of clinical application research.

Therefore, this paper sorts out these confusing problems, throws bricks and stones, and hopes to attract everyone's attention and jointly promote the study
of the immune characteristics and functions of MSCs.

-01-Mesenchymal stem cells are not equivalent to immunosuppressants

Most of the animal experiments and in vitro experimental data show that MSCs have inhibitory effects on T cells, B cells, NK cells, DC cells, and macrophages, and promote Treg cells
.
That is, in general, MSCs have a negative effect on the immune system, inhibit the immune response and induce immune tolerance
.
If this theory is correct, then MSCs are almost equivalent to immunosuppressants, and the clinical application of MSCs will increase the risk of infection and the risk of high tumor incidence
.

In fact, large clinical studies have demonstrated the safety of
MSCs treatment.
A number of clinical trials have found no difference in infection rates between MSCs-treated and control groups, and it is clearly stated that MSCs treatment does not increase the risk of infection, and there is no difference
in cachexia and tumorigenesis between MSCs-treated and control groups.
Long-term observation of clinical trials has also not found an increase in infection and tumorigenesis of MSCs
.
The team of Fuzhou General Hospital in China published an article in the top international journal JAMA, confirming for the first time through randomized controlled clinical trials that autologous BMSCs exert immunosuppressive effects after solid organ kidney transplantation, and play a therapeutic effect
similar to immunosuppressants.
At a one-year follow-up period after transplantation, kidney transplant patients in the MSCs-treated group had a lower risk of acute rejection and infection, while promoting better recovery
of kidney function.

Therefore, MSCs are not equivalent to immunosuppressants
.

Since the new crown epidemic, China and many countries have successively carried out clinical studies on the treatment of COVID-19 by MSCs, which has preliminarily proved that intravenous infusion of MSCs for the treatment of COVID-19 is safe and effective, especially to reduce the mortality rate of critically ill patients and promote the recovery
of lung function.

In May 2020, the Mayo Clinic in the United States found a trend
towards improvement in radiological outcomes, lung function (lung compliance, tidal volume, PaO2/FiO2 ratio, alveolar capillary damage), and inflammatory biomarker levels in COVID-19 patients treated with MSCs.
Even in the presence of the new coronavirus in COVID-19 patients, the use of MSCs improves the clearance of the new coronavirus by the body's immune cells, and promotes the resolution of inflammation and the recovery
of damaged tissues.

In summary, intravenous infusion of MSCs did not suppress the immune response and did not impair immune clearance (which is important).

Therefore, MSCs are not different from immunosuppressants
.
A growing body of data demonstrates that MSCs can be an important weapon for host-directed therapies to improve host cell clearance responses to pathogens and prevent or reduce antibiotic resistance waits
.

-02-Mesenchymal stem cells have a positive effect on immunity

MSCs are co-cultured with T cells, and the activation inhibition of T cells by MSCs is temporary; If the MSCs are removed, the T cells can proliferate
again in response to antigen stimuli.
During viral infection, MSCs do not inhibit CD8+ T lymphocyte function, but promote CTL response
by releasing IFN-γ.

Clinical studies of MSCs in patients with drug-resistant tuberculosis were conducted in Belarus, and clinical data proved that MSCs treatment can greatly improve the prognosis of patients with MDR-TB, with 75% of patients cured
.
Interestingly, after the infusion of MSCs, most of the immune cells of tuberculosis patients produced more IFN-γ under antigen stimulation, suggesting that the function of immune cells was restored to a certain extent, and there was no immunosuppressive effect
.

MSCs can upregulate the number of Treg cells, thereby exerting a protective effect on the heart in inflammatory cardiomyopathy
.
A large sample of clinical studies in China has found that Treg cytopenia is common in patients with rheumatoid arthritis, and after MSCs treatment, patients with increased Treg cells have obvious relief of RA-related clinical symptoms and signs, and the efficacy of MSCs is well maintained
.

Macrophages are very critical immune system players, playing a role
in tissue repair, homeostasis, and clearing bacterial infections.
Under the action of cytokines secreted by MSCs, the phagocytic and killing ability of monocytes/macrophages and neutrophils is enhanced
.

After treatment with MSCs, macrophages in infected tissues showed a predominantly M2 phenotype, while infected tissues not treated with MSCs mainly contained M1 macrophages
.
MSCs promote enhanced phagocytosis and mainly regulate PGE2 and phosphatidylinositol 3-kinase, which are effective in promoting macrophage phagocytosis to unconditioned bacteria
.
MSCs can also directly deliver mitochondria to macrophages through the structure of nanotunnel tubes, thereby improving the phagocytic ability
of macrophages.
MSCs enhance the phagocytic activity of macrophages, which relies in part on the uptake
of extracellular vesicles containing MSCs' mitochondria.

MSCs have a strong function of supporting hematopoiesis, that is, MSCs can promote the better differentiation and development of hematopoietic stem cells into various blood cells, including various immune cells, so MSCs are not all negative effects (inhibition)
on the overall immune system.

-03 - Whether direct contact is required for immunomodulation

There is also an idea that cell-to-cell binding contacts through adhesion molecules (ICAM-1 and VCAM-1) are precursor conditions for MSCs to exert immunosuppressive effects
.
Italian scientists were the first to prove that MSCs inhibiting the proliferation of T cells does not need to rely on the contact
between cells.
Subsequently, different laboratories in the United States, Italy and Germany used transwell experimental studies to prove that MSCs inhibit the activation of T cells without the need for
contact between cells.
Soluble inducible factors

Since the immunosuppressive function of MSCs does not need to rely on the contact between cells, soluble factors should exist to inhibit T cell activation
.
So, what are the soluble factors?

From the earliest HGF and TGF-β, to IDO, NO, HO-1, PGE2, HLA-G5, Galectin-1 and Sema-3A, Galectin-9, etc
.
If these factors are all involved, what are the commonalities behind these factors?

In 2006, it was shown that IFN-γ induced MSCs to express IDO, thereby enhancing the inhibitory ability
of MSCs on T cells.
There are also experimental data that finding that blocking IDO does not reverse the inhibitory effect
of MSCs on T cells.
The Irish research team found that mouse BMSCs do not express PD-1 or PD-L1/PD-L2, IDO, but IFN-γ can stimulate MSCs to express PD-L1 and IDO, thereby enhancing the immunosuppressive function
of MSCs.

In 2007, the team of Professor Katsutoshi Ozaki in Japan found that the immunosuppressive ability of NOS-/mouse-derived BMSC decreased and could not significantly inhibit the proliferation of T cells; The interaction between MSCs and T cells induces iNOS expression of MSCs and increases the production of NO, thereby inhibiting the proliferation
of T cells.
Chinese scientists further proved that mouse BMSCs knocked out iNOS, or MSCs knocked out IFN-γ receptor 1, both lost immunosuppressive ability and could not effectively treat GVHD
in mice.
However, another 2007 article demonstrated that IFN-γ receptor 1-deficient MSCs do not express IDO, and that the effectiveness of MSCs in inhibiting PBMCs (mostly immune cells) is not affected
by IFN-γ receptor 1.
Who is right and who is wrong?

TNF-α and IFN-γ jointly stimulate BMSCs, inducing MSCs to express iNOS, thereby producing NO, which induces lymphocyte apoptosis
.
Subsequently, it was found that TNF-α and IFN-γ jointly stimulated BMSCs, inducing MSCs to express the NO-produced by iNOS, which in turn could also induce apoptosis
of MSCs.
The data results of these experimental studies show that NO, as a small molecule of vasodilation, can induce apoptosis of lymphocytes and BMSCs, impairing the immune system and the
body's ability to repair.
If this theory holds, then a related drug (Viagra) developed based on the second messenger NO small molecule may damage the immune system and repair ability (so scary).

In fact, this experiment lacks data
on the mechanism of action of NO-induced apoptosis of MSCs.

In addition, there is no binding point of TNF on the surface of MSCs cells, so why are there still data to prove that TNF can have a damaging effect on MSCs? In a similar situation, MSCs do not express the receptor (CD14) of LPS, so why can we still see experimental data on the effect of LPS on MSCs? One of the two explanations for these experimental data is the need to demonstrate that TNF-α and LPS can induce the expression
of TNF membrane receptors and LPS receptors of MSCs, respectively.

At present, there is no experimental evidence that TSG-6 secreted by MSCs directly inhibits T cell activation, but more experimental data prove that TSG-6 has anti-inflammatory effects
.
TSG-6 secreted by MSCs also polarizes macrophages into an anti-inflammatory M2 phenotype
.
In flat dish cultures, MSCs do not express TSG-6, whereas when MSCs are cultured in 3D spheroids, TSG-6
is expressed at high levels.
However, many teams have shown that MSCs derived from bone marrow, amniotic membranes, fat, and umbilical cord blood express TSG-6, which mediates anti-inflammatory protection
.
Mouse and human MSCs with reduced TSG-6 expression also had a weakened
immunosuppressive capacity.
Therefore, it has been suggested that TSG-6 can be used as an indicator
of the immunosuppressive ability of response MSCs.
However, there are also privately communicated data suggesting that MSCs express very low levels of TSG-6, but still have strong immunosuppressive function
.

Soluble IL-2R is an easily overlooked soluble factor
that inhibits T cell activation.
Co-culture experiments of MSCs and CD4+ T cells found high expression of IL-2R, which can neutralize IL-2
secreted by T cells.
As early as 2002, Professor Bartholomew's team in the United States reported that MSCs have the function of inhibiting lymphocyte activation, and in vitro experimental MSCs and T cell co-culture, the addition of IL-2 can partially reverse the inhibitory effect
of MSCs.
This suggests that when the activation power of IL-2 is greater than the inhibitory effect of MSCs, the ultimate effect is to promote the proliferation
of T cells.

It has been suggested that MSCs in humans, monkeys, and pigs exert immunosuppressive effects by expressing large amounts of IDO, while rodent and rabbit MSCs exert immunosuppressive effects
by expressing large amounts of iNOS.
But this view and supporting data do not explain the phenomenon of human MSCs inhibiting animal immune cells (cross-species inhibition), so this view is very debatable
.

-04 - Whether it has antigen presentation function

As early as 2003, the team of Professor William T.
Tse of Boston Children's Hospital in the United States confirmed that MSCs do not have the function of antigen-presenting cells (APCs), and MSCs cannot induce apoptosis
of T cells.

Multiple animal experiments have found that the allogeneic MSCs after infusion are not complete immune escape, but have certain antigenic characteristics, which can induce the body to produce immune memory, including memory CD4+ T and memory CD8+ T cells; After allogeneic MSCs are infused back into the body, they can still be recognized and eliminated by the immune system, resulting in the treatment effect of allogeneic MSCs being slightly less effective than autologous MSCs
.
A single injection of MHC-mismatched umbilical cord MSCs does not induce an immune response, but MSCs are immunogenic
when injected at the site of inflammation, repeated in the same area, or stimulated with IFN-γ prior to injection.
Therefore, some experts have proposed that MSCs do not have immune immunity characteristics, but only have low
immunogenicity.

MSCs themselves have low immunogenicity, can be used allogeneically or across species, do not cause immune rejection, but the body's clearance of MSCs accelerates
.
Although MSCs inhibit the activation of T cells, MSCs can promote the survival of T cells, leaving T cells in a resting state
.
MSCs can secrete a large number of nutritional factors, have the function of nutritional support, promote the repair of injured tissue cells, and also promote the survival
of T cells.

It should be noted that if the purity of the cultured MSCs is not enough (BMSCs are more common), then the residual heterogeneous cells increase the immunogenicity of the overall cultured cells, resulting in the immune rejection of the body after these cells enter the body
.

How to judge the purity of MSCs can currently be based on the results
of flow cytometry detection of cell phenotypes.
In 2006, the International Association of Cell Therapy proposed the identification criteria for MSCs, as follows: -05-IFN-γ What is the true effect on MSCs

In 2003, researchers found that IFN-γ stimulated MSCs to express MHC-II, but MSCs did not function
as antigen-presenting cells (APCs).
IFN-γ stimulates MSCs to express MHC-II, an effect that can be inhibited
by TGF-β.
Interestingly, MSCs themselves secrete very low concentrations of IFN-γ, but high concentrations of TGF-β
.

Because MSCs do not express MHC II and express MHC I low, the immunogenicity of MSCs is weak
.
If MSCs are stimulated with IFN-γ, MHC I and MHC II can be upregulated, which may predispose MSCs to rejection in immunocompetent hosts
.
MSCs with high expression of MHC-I (IFN-γ promotes the expression of MHC-I molecules), MSCs are easily killed by CD8+ CTL cells
.
If the cell surface of MSCs is low or does not express MHC-I, MSCs are easily recognized and killed
by NK.

In fact, whether it is allogeneic or autologous, MSCs are transient escapes the surveillance of the immune system, and are eventually recognized by the immune system, but allogeneic is easier to be recognized and eliminated
by the immune system than autologous.

In 2006, researchers found that low levels of IFN-γ increased MHC-II expression in MSCs, but as IFN-γ levels increased, MSC MHC-II expression gradually decreased
.
However, through the data analysis of the article, it was found that the BMSC used in the experiment, under normal culture, MSCs have high expression of MHC-II (at least 80%-90%), then, these cells are no longer MSCs, MSCs themselves are low expression MHC-II (no more than 2% HLA-DR).

It is strange that this article was published, and many experts agree with the data and conclusions
of this article.
Could it be that these experts have forgotten the identification criteria for MSCs? There is also an idea that the expression of MHC II is not necessarily related
to the efficacy of MSCs.

It is necessary to be cautious about these experimental results and views, MSCs have high MHC II expression, that is, the immunogenicity of MSCs is increased, then the consequence is that MSCs are cleared more quickly by the body, so that they cannot better play a therapeutic role
.

-06-Other problems with mesenchymal stem cell immunoregulation

▉ MSCs with dexamethasone

Dexamethasone can inhibit the proliferation of T cells and induce apoptosis of T cells, and MSCs can also inhibit the proliferation of T cells, but interestingly, dexamethasone combined with MSC, dexamethasone can promote the proliferation
of T cells by inhibiting phosphorylation of STAT1 and inhibiting the expression of iNOS by MSCs.

This negative negative positive phenomenon can only be explained by the fact that when dexamethasone faces MSCs and T cells at the same time, it can only act on MSCs preferentially, leaving T cells alone
.
MSCs and inflammation levels

In 2013, the international journal Cell Stem Cell had an article arguing that MSCs promote the activation of T cells in a low-level inflammatory (mildly inflamed) environment, which will aggravate inflammation; However, MSCs can only induce the proliferation of Treg cells to eliminate inflammation in an environment with high levels of inflammation (severe inflammation
).
This article and another hypothesized that while MSCs have immunosuppressive effects in the case of strong inflammation, weak inflammation causes MSCs to enhance the immune response
.

Translated into layman's terms, that is, in a mildly inflammatory environment, MSCs dislike that the inflammation is not serious enough, and they must first transform into pro-inflammatory cells to make mild inflammation progress to severe inflammation
.
MSCs can then exert anti-inflammatory effects and eliminate inflammation
.
This view is magical!! Is that right? Then look below
.

A very simple experiment can refute this theory, that is, to establish a mouse model of mild liver injury, and then give MSCs treatment; If after treatment with MSCs, these mice with mild liver injury progressed to severe liver injury (severe hepatitis), then this theory holds; If the liver function of mice with mild liver injury improves after MSCs treatment, then this theory does not hold
.

Dear hands-on students, you can try, this is a great opportunity
to do experiments with your own hands to challenge the theory of big coffee.
It should be noted that it is necessary to clarify that the cells used for the treatment of MSCs are indeed not MSCs of poor purity and cell quality

Organizational heterogeneity of MSCs

Studies have suggested that the immunomodulatory ability of umbilical cord and amniotic MSCs is significantly better than that of bone marrow and cord blood sources
.
The immunomodulatory ability of fat-derived MSCs is also significantly better than that of bone marrow
.
However, a recent study showed that fat-derived MSCs were slightly more immunosuppressive than bone marrow from the same donor, but there was no statistically significant difference
.
This result is consistent with mouse experiments, that is, there is no difference
in the immunosuppressive ability of fat-derived and bone marrow-derived MSCs derived from the same mouse to inhibit T cell proliferation.
However, there is no clear relationship
between immunomodulatory and proliferative potential of MSCs.
Therefore, it is not clear whether
the immune regulation ability of MSCs from different tissue sources is strong.

-07 - Summary at the end of the article

In short, we first need to clarify the various factors affecting the immunomodulatory ability of MSCs, and then screen out the subsets of MSCs with strong immunomodulatory ability, or pretreat with relevant factors to strengthen their immunomodulatory ability before infusion, and finally use them for indications suitable for immunomodulation (such as GVHD, systemic lupus erythematosus, rheumatoid arthritis, etc.
).

What do you think of the mixed experimental results and expert opinions? The author believes that if the theories obtained by cell experiments and animal experiments cannot explain clinical phenomena (results), then what needs to be considered should be the theories
obtained by current cell and animal experiments.