**4. Mechanism of TEX-mediated immune suppression**

Immunostimulatory TEX from tumors may spread to distant tissues and organs, impairing systemic anti-tumor immune responses. Signaling molecules in the tumor microenvironment aid in tumor development and inhibits the immune response, with T cells being highly vulnerable to TEX-driven negative messages [7, 43]. Unlike other leukocytes which engulf other cells, T cells interact with the TEX using the ligands and surface receptors. It leads to the signal-driven influx of Ca2+ in the cells and activation and suppression of pathways along with downstream modified responses. Two major receptors on the T cells that are negatively regulated by the TEX are the interleukin 2 receptor (IL-2R) and T cell receptor (TCR) [44]. The TCR zeta chain is downregulated consistently on the incubation of TEX with the T cell. In addition, JAK expression and phosphorylation are diminished, which is responsible for the production of IL-2, IL-7, and IL-15, all of which are deleterious to T cell proliferation. Further, TEX downregulates the proliferation of CD4+ T cells but upregulates the expansion of CD8+ T cells [45]. However, in the case of normal cell-derived exosomes, the proliferation of all T cells is experienced [46]. Also, TEX regulates the STAT5 function in the T cells, as STAT5 phosphorylation in the case of CD4+ T cells increases while the phosphorylation of STAT5 in CD8+ T cells decreases [47]. The level of CD69 on the surface of CD4+ T cells is also reduced due to TEX immunosuppressive function.

T cell inhibitory and apoptotic receptors are directly engaged by tumor-derived exosomes (TEX). To enhance T cell death, TEX expresses Fas ligand (FasL), TNFalpha, TRAIL, and Galectin-9 that interacts with counterpart T cell receptors like FAS, TNF R1, TRAIL R2, and Tim3 [48]. The TEX is also found to inhibit the antigenspecific T cell, as in the case of human melanoma, wherein specific T cells are generated via the melanoma-specific pulsing of the DC cells. Also, evidence suggests the presence of membrane-bound FasL and PD-L1 on TEX [49]. TEX-mediated apoptosis of CD8+ T cells is associated with canonical features like the caspase 3 cleavage, annexin V binding, loss of mitochondria membrane potential, DNA fragmentation, and cytochrome C release [45]. It suggests the involvement of extrinsic and an intrinsic mechanism for the cascade of apoptosis of the CD8+ T cells. Also, the PI3K/ AKT pathways are the main target for TEX-driven apoptosis in CD8+ T cells: AKT dephosphorylation in a time-dependent manner decreases the expression of BCL-xL, BCL-2, and MCL-1 along with the increase in BAX was observed when TEX was incubated with CD8+ T cells [50]. All these data indicate that TEX may not be internalized by the T cells; instead, the negative signaling by the surface receptors modulates the function of T cell response. Also, TEX-driven transcription changes are regulated by the type of recipient cell, activation status, and presence or absence of exosomes. TEX is also responsible for the apoptosis of CD8+ T cells with the help of death ligands and interaction via the Fas/FasL pathway [51]. Also, it has been shown in the lymphoma animal model that TEX protects cancer cells from complement-dependent cytolysis by binding to the complements [52].

#### *Tumor-Derived Exosome and Immune Modulation DOI: http://dx.doi.org/10.5772/intechopen.103718*

Cancer cells upregulate PD-L1 to avoid immune identification by causing anergy in PD-1+ T cells. Although immune checkpoint inhibitors have shown extraordinary effectiveness, most patients do not react to PD-1/PD-L1 inhibition. Paracrine immunosuppression may now include recently discovered exosomal-PD-L1 as well as cell-to-cell interaction [53]. By both direct and indirect means, exosomes seem to be capable of immunomodulating PD-L1 (**Figure 3**). Recent research shows that PD-L1 is active on exosome membranes and may promote tumor development by inhibiting CD8+ T cell proliferation and cytotoxicity [53]. Exosomes from human breast cancer cells contain PD-L1, while PD-L1 knockout (KO) cells do not [54]. In a unique experiment, two groups of mice were treated with exosome-expressing PD-L1FLAG and exosome-expressing no PD-L1 (PD-L1-KO). Animals treated with exosomeexpressing PD-L1FLAG showed considerably larger tumor volumes than mice treated with exosome-expressing no PD-L1, demonstrating that exosomal PD-L1 stimulates breast cancer tumor development. Exosomes expressing PD-L1 dramatically reduced T cell death, demonstrating that PD-L1 signaling may limit T cell killing of cancer cells [55]. Exosomes isolated from the WM9 and WM164 human melanoma cell lines express PD-L1, and may function as an anti-PD-L1 antibody sink [34, 38]. Chen et al., also reported exosomal PD-L1 in human non–small cell lung cancer (NSCLC) and breast cancer cell lines. Recent findings suggest that PD-L1 exosome's immunosuppressive effects are not limited to the tumor microenvironment, and that exosomes might cause systemic alterations in adaptive immune components (**Figure 3**) [49]. Functional modifiers that stimulate PD-L1 expression in target cells might be delivered via tumor-derived exosomes. T cell dysfunction may be caused directly by PD-L1+ monocytes or macrophages as a result of a PD-L1-PD-1 interaction [56].

#### **Figure 3.**

*TEX and PD-L1: By activating the PD-L1/PD1 axis, cancer cells adapt and exploit the immune system to elude immune monitoring. a. Exosomal-PD-L1 interacts with T-cells through paracrine signaling, inactivating T cell effector activity. b. Induction of functional PD-L1 in target cells by exosome cargo (miRNA). The PD-L1 on the surface then interacts with T cells, inactivating effector activity.*

Further investigation is needed regarding exosomal PD-L1 and exosome-induced PD-L1 in immune modulation and cancer. Exosomal-PD-L1-based paracrine immune regulation may help create novel therapeutic options.

Another interesting aspect of TEX-mediated immunomodulation involves the anti-phagocytic surface proteins called 'do not eat me' signals, such as the cluster of differentiation (CD) 47 help healthy and normal cells evade macrophage-mediated phagocytosis, while a loss of 'do not eat me' signals in apoptotic or senescent cells leads to their systemic clearance [57]. Cancer cells use a similar technique to avoid macrophage-mediated clearance by overexpressing 'do not eat me' signals on their surface. TEX overexpressing CD47 renders phagocytic inactivation by interacting with SIRPα of immune cells and thereby enjoys prolonged circulation [58]. Downregulation of CD47 expression or inhibition of CD47-SIRPα can be an interesting approach to activate cancer cell/TEX phagocytosis [59].
