**4.2 Exosomes and immune cells**

*Immune Response Activation and Immunomodulation*

and Treg responses [69–71].

from degradation.

many more [78].

**4.1 Exosome formation to secretion**

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

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

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

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

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

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

**114**

well [77].

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 studied for their therapeutic potential in various diseases.

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 molecules and their target effect.

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

exerted an inhibitory effect on the differentiation of activated T cells, reduced T-cell proliferation, and IFN-γ secretion in an in vitro stimulatedT-cell model [93]. Favaro et al. has shown the effect of BM-MSC-derived exosomes on PBMNCs isolated from type I diabetic patients. These exosomes were able to inhibit the IFN-γ production and significantly increased the production of immunomodulatory mediators such as PGE-2, TGF-β, IL-10, and IL-6 [94]. The in vitro studies were complemented by the in vivo studies which confirmed the immunosuppressive effect of exosomes in mouse allogeneic skin grafting models [95]. Bai et al 2017 have subcutaneously administered exosomes isolated from human embryonic stem cellderived MSCs and showed that there was delayed occurrence of GvHD for 2days, concomitant with increasing Treg polarization. In continuation, the author has also demonstrated that exosomes released from WJ-MSCs can effectively ameliorate experimental autoimmune uveoretinitis (EAU) in rats by inhibiting the migration of inflammatory cells [95]. Moreover, there is only single case study on humans in which MSC exosomes have been tested in the treatment of resistant grade IV acute GvHD patient which experienced improvement in symptoms for 5 months. There were no side effects reported, and the decrease of pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ was observed. The anti-inflammatory molecules IL-10, TGF-β1, and HLA-G contained in the exosome preparations were believed to contribute to the immunosuppressive effect of MSC-Exo [96].

Although there are limited studies available using MSC-derived exosomes, future advancements into research and gain in in-depth knowledge of immunomodulatory properties of these MSC exosomes could be seen. These nano-vesicles can be developed as cell-free therapies. The use of these exosomes as cell-free therapies provides following key advantages:


Despite these advantages of MSC-derived exosomes, there has been a lack of manufacturing process that is required to generate exosomes with clinically relevant quantities. Therefore, there is an urgent need for technological advancements. Nevertheless, regulatory requirements will be necessary to establish the safety and efficacy profile of these exosome products.
