*3.7.2 MSC interaction with NK cells*

*Immune Response Activation and Immunomodulation*

In addition to the above molecules, several additional mediators are produced by MSCs or other adult stem/progenitor cells upon inflammatory stimulation, such as the inhibitory surface protein programmed death ligand 1 (PD-L1) [54], heme oxygenase-1 (HO-1) [55], leukemia inhibitory factor (LIF), galectins [56]*,* and TGF-β [57]. However, their modes of action and underlying molecular mechanisms that drive MSC-mediated immunosuppression require further investigation.

The antigen-specific immune system allows the development of immunological memory. It comprises of CD4+ T helper and CD8+ cytotoxic T lymphocytes that deliver a customized antigen-specific immune response following antigen processing and presentation by antigen-presenting cells (APCs). T helper cells comprise a subpopulation of cells called Tregs, which are specialized in suppression of T cell-mediated immune response [58]. The innate immune system plays an important role in the activation and subsequent course of adaptive immune response [59]. In addition, MSCs are able to suppress in vitro T-cell proliferation induced by cellular or non-specific mitogenic stimuli through the secretion of various soluble factors that include (transforming growth factor-beta 1) TGF-β, HGF, PGE-2, IDO, HLA-G5, and NO. The effect of these suppressive factors is upregulated by pre-sensitization of MSCs with TNF-α and IFN-γ. It is also known that MSCs polarize T cells toward a regulatory phenotype that serves as an important mechanism by which MSCs dampen inflammation [60, 61]. Tregs comprise a subpopulation of T helper cells, which are specialized in suppression of T cell-mediated immune response and characteristically express the forkhead box P3 (Fox P3) transcription factor. These are two main subsets of Tregs including a population of Fox P3+ natural Tregs which are thymus derived and specific for self-antigen and induced or adaptive Tregs that are derived from mature CD4 + CD35-FoxP3 precursors in the periphery following inflammatory stimuli. The in vitro co-culturing of MSCs with PBMNCs induced the differentiation of CD4+ T cells into CD25 + FoxP3+ expressing regulatory T cells [40, 62]. The possible reason of abovementioned mechanism is due to cell-cell contact of MSCs with helper T cells and secretion of PGE-2 and TGF-β. All together, these studies indicate that MSCs are able to maintain the balance between

**3.7 Molecular and cellular interaction of MSCs with innate and acquired** 

inflammatory effector T cells and anti-inflammatory Tregs.

B cells are also a major cell type involved in adaptive immune response, known

for antigen presentation and antibody production. The balance between the different B-cell subsets has been identified as an important factor for optimal graft outcomes. To support the beneficial effect of B-cell depletion at the time of transplantation to impair T cell-mediated allo-response, the CD8 and CD4 T-cell memory is impaired when the antigen-presenting function of B cells is absent [63]. The exposure of enriched B-cell population to irradiated third party PBMNCs led to an increase in immunoglobulin (Ig) production that was abrogated by the addition of MSCs. There are diverse results among the studies to analyze the effect of MSCs upon exposure of isolated pure B cells [64]. These effects have been shown to be cell-cell contact independent or indirect through inhibition of pDC-induced B-cell maturation. On exposure, MSCs increased the viability of B cells and mediated the arrest of cell cycle at G0/G1 and inhibition of their differentiation into plasma cells and subsequent Ig formation, whereas it was observed that pre-treatment of

**3.6 Other mediators**

**immune cells**

*3.7.1 MSC interaction with T and B cells*

**112**

Natural killer cells or NK cells are a type of cytotoxic lymphocytes critical to the innate immune system, evolve as progenitors in the bone marrow, and circulate as mature cells in the blood. They provide rapid responses to viral-infected cells, acting 3 days after infection, and respond to tumor formation. They play a major role in the mechanisms of rejection of graft and are central to the regulation of cytotoxicity in response to human leukocyte antigen molecule. With increasing trends in therapeutic usage of MSCs for treatment of GvHD, it is important to investigate the underlying effects of interaction of MSC and NK cells. They function in the manner that they get activated and inhibited on cell surface because of receptors transmitting the signal into the cell. Usually, NK cell possesses regulatory functions and can secrete cytokines and chemokines which modulate the host's immune response. IL-12 is the most important pro-inflammatory factor which responds to penetrating pathogens and acts through its high affinity receptors. It is released from accessory cells like monocytes, macrophages, and dendritic cells (DCs). Also, the most important cytokine released by NK cells is IFN-γ which is produced upon stimulation of IL-12. The NK cell-derived IFN-y reinforces the expression of IL-12 and DCs via feedback mechanism. BM-MSCs directly interfere with the proliferation, cytokine production, and in some cases cytotoxicity of NK cells. MSC-NK interactions are complex and largely dependent on the microenvironment and activation status of the NK cells. Mainly, MSCs suppress the production of IL-2, IL-15, and INF-γ but not the cytotoxicity of freshly isolated NK cells. In addition, when activated NK cells come into contact with the MSCs, it interferes with NK-mediated cytotoxicity which is primarily mediated by cell-cell contact and secretion of IDO, PGE-2, TGFβ1, and HLA-G5. Other reports mentioned that when licensed MSCs were exposed to IFN-γ, they are protected from NK-mediated cell killing, potentially due to their upregulated cell surface expression of HLA-I and downregulation of ULBP-3. This alongside an increased production of both IDO and PGE-2 offers multiple mechanisms for dampening NK responsiveness to the MSCs [59, 67, 68].

### *3.7.3 MSC interaction with dendritic cells*

The potent antigen-presenting cells (APCs) and dendritic cells (DCs) play a pivotal role in initiating immune response. The life span of DCs can be divided into two major phases, an immature stage and a mature stage. These phases can be differentiated further on the basis of molecules expressed (CD80, CD86, OX62, HLA-II, and CD11b/c) on their surface. DCs can be immunostimulatory or immunosuppressive,

depending upon their maturation stage and specific DC subset. Immature DCs (iDCs) express low levels of HLA-II but no co-stimulatory molecules. The interaction of MSCs with DCs leads to the inhibition of maturation of monocytes and CD34+ precursor cells. Moreover, the direct activation of DCs leads to the release of PGE-2, IL-6, TSG-6, MCSF, and jagged-2 mediated signaling. Tolerogenic phenotype occurs when DC secretome of pro-inflammatory cytokines (TNF-α and IL-12) shifts toward anti-inflammatory IL-10 in which further downstream induces Th2 and Treg responses [69–71].

## **4. Paracrine interaction with in the niche (exosomes)**

Advances in stem cell technology have opened interesting perspectives within the realm of regenerative medicine. As reported, MSCs participate in repair and regenerative processes via different mechanisms like homing and transdifferentiation and immunomodulation, which depends on paracrine mechanisms [72, 73]. The initial studies using MSCs were based on local engrafting of MSCs and differentiating into multiple tissue types. However, with the in-depth study of different mechanisms of MSC action, it has been reported that <1% MSCs are able to survive transiently after systemic administration [74]. This suggests that paracrine mechanisms through secretion of various molecules called secretome might be the possible mechanism for MSC regenerative potential. This has attracted significant attention for the potential use of MSC secretome in tissue repair and regeneration.

The secretome released by MSCs includes various biologically active growth factors and cytokines which aid in immunomodulatory properties of MSCs [75]. However, we cannot neglect the fact that the secretome released in the milieu of ECM of the cells is an easy target for denaturation due to the presence of proteases and other enzymes in the microenvironmental niche. Therefore, these growth factors and cytokines have been shown to be packed into small vesicles called exosomes which are secreted by MSCs in the extracellular milieu of cell along with the secretome [76]. They function through encapsulation of biological active molecules such as miRNA, proteins, and immunomodulatory molecules and protect them from degradation.

#### **4.1 Exosome formation to secretion**

Exosomes are lipid membrane-bound extracellular vesicles which possess a diameter of 30–120 nm and a density of 1.09–1.18 g/mL and are secreted by all cell types. These exosomes carry cellular components like proteins and nucleic acids and aid in cell-cell communication. The exosome was first discovered in 1984 by Johnstone in sheep reticulocytes [77]. It was initially believed that exosomes remove unwanted proteins from cells. Later on, it was demonstrated that many other cell types also secrete exosomes including immune cells, cancer cells, stem cells, and many more [78].

Exosomes are endosomal in origin, formed within multivesicular endosomes (MVEs). These vesicles are being released when membranes of MVEs fuse with that of the cellular plasma membrane. These exosomes express various surface markers like CD63, CD81, and CD9. They carry surface molecules that are present on the parent cell which aids in identification of exosomes and their parent cell source as well [77].

Moreover, the exosomes secreted by stem cells carry various proteins (growth factors and cytokines) and nucleic acids (mRNA and miRNA) that can influence their mode of action [79]. The content carried by these vesicles depends on the type

**115**

*Mesenchymal Stem Cells Modulate the Immune System in Developing Therapeutic Interventions*

of cell (including source of cells) and its state of activation. Once released, these vesicles have local as well as remote effect by interacting with the neighboring cells

The exosomes were studied for their multifaceted application in antigen presentation, and vastly studied immune cell was dendritic cells. The clinical studies have been conducted to evaluate the dendritic cell derived exosomes for their therapeutic potential. However, compared to preclinical studies, only a few clinical trials have been conducted using exosomes. Reported studies were conducted where dendritic cell-derived exosomes were evaluated for their safety, tolerability, and efficacy in cancer patients. Exosomes carry parental cell surface marker expression. In this regard, DC derived exosomes are HLA-II positive as a result they can only be used in patient specific studies [80, 81]. In contrast, MSCs are immunologically naïve as they express only HLA class I molecules and lack HLA class II, CD40, CD80 and CD86 expression on their cell surface. Also, they are capable of immune escape and fail to induce an immune response by the transplanted host. Similarly, exosomes secreted by them are also immunologically naïve [82]. Considering all of the above properties, several recent studies have focused their research on evaluation of stem cell-derived exosomes in the area of immunomodulation with fewer reports.

In a recent study, exosomes derived from MSCs were specifically identified to mimic the effect of MSCs, and this paved the way to cell-free therapeutic approach using exosomes instead of the cell itself [83]. The first report using MSC exosomes were in cardiovascular diseases where Lai et al. [85] identified exosomes as the cardioprotective components in MSC paracrine secretion [84]. This was followed by several other studies where exosomes isolated from tissue-specific MSCs were

Initial studies were performed on bone marrow-derived exosomes for evaluating their regenerative potential in cardiovascular diseases [85], acute kidney injury [86], bone defects, etc. [87]. By 2013, only researchers started exploring the regenerative potential of exosomes derived from adipose tissue and Wharton's jelly sources. These studies have explored the various mechanisms by which these exosomes mimic MSCs. The content of these exosomes was evaluated by using various techniques like RNA sequencing, mass spectrometry, etc., to identify different

Conforti et al. reported the effect of MSC-derived vesicles on B-cell proliferation which was further confirmed by Di Trapani's group in 2016 [88, 89] . They observed that exosomes had higher levels of miRNAs compared to MSCs and induce inflammatory priming via increasing levels of miR-155 and miR-146. These are two miRNAs involved in the activation and inhibition of inflammatory reactions. Similar studies were reported where MSC-derived exosomes were shown to increase the ratio between regulatory and effector T cells along with the increase in cytokine such as IL-10 [90]. Similarly, Chen et al*.* [91] has also reported immunomodulatory effects of MSC-derived exosomes toward peripheral blood mononuclear cells (PBMNCs) focusing specifically on T cells. It was observed that there was significant inhibition of pro-inflammatory cytokines, IL-1β and TNF-α, but enhancement of the expression of anti-inflammatory cytokine, TGF-β1. This cytokine profile in their study mimics the immunomodulatory effect of MSCs [91]. Zhang et al. showed that these exosomes may polarize monocytes toward M2-like phenotype, which in turn induces CD4+ T-cell differentiation into regulatory T cells [92].

Blazquez et al. demonstrated AD-MSC-derived exosomes as a therapeutic agent for the treatment of inflammation-related diseases. They showed that exosomes

studied for their therapeutic potential in various diseases.

molecules and their target effect.

or by circulating in the body fluids (bloodstream, saliva, serum, etc.).

*DOI: http://dx.doi.org/10.5772/intechopen.80772*

**4.2 Exosomes and immune cells**

of cell (including source of cells) and its state of activation. Once released, these vesicles have local as well as remote effect by interacting with the neighboring cells or by circulating in the body fluids (bloodstream, saliva, serum, etc.).
