The effects of tumor‐derived exosomes on T‐cell function and efficacy of cancer immunotherapy

Tumor‐derived exosomes (TEXs) are a class of extracellular vesicles which play an important role in the tumor microenvironment. These vesicles have multiple biological functions including promotion of cancer progression and reduction of anti‐tumor immunity. Recently, interaction between TEXs and immune cells are of great interest in cell‐based immunotherapy. Here, we review the effects of TEXs on the survival and functions of T cell subsets, as well as their clinical applications. Unraveling the immunoregulatory function of exosomes allows a better understanding of the molecular and cellular basis for cancer immunotherapy.


INTRODUCTION
Exosomes are a special group of extracellular vesicles (EVs) of 30∼150 nm in diameter and released by almost all cells. 1 In the early 1980s, Johnstone et al 2 first discovered that some small vesicles loaded with transferrin receptors were released by the reticulocytes of sheep during their maturation. At one time exosomes were considered as "cell garbage collectors" to dispose of the cellular waste. Today, exosomes have been emerging as communication vehicles to transfer information between cells, and play a critical role in both health and disease. [3][4][5] heterogeneous carriers, containing various inhibitory lipids, proteins and nucleic acids, which have shown potential regulatory functions in immunotherapy. Since T lymphocytes are the major immune effector cells in anti-tumor immune responses, here we mainly focus on the effects of TEXs on T lymphocyte subsets, including CD4 + , CD8 + and Treg cells, and review the complex mechanisms underlying the crosstalk between TEXs and T cells.

Three modes of interaction between TEXs and T cells
Generally, there are three pathways for TEXs to transfer information: (1) TEXs deliver intercellular signal through receptor-ligand binding; (2) TEXs fuse with the membrane of recipient cells and release their "cargo"; (3) Recipient cells can phagocytose and internalize TEXs. 10 In fact, how TEXs interact with T cells is still under debate. Muller et al. 11,12 found that little PKH26-labeled TEXs are internalized by T cell subsets following 48-72 h co-incubation, suggesting that the receptor-ligand interaction alone is sufficient to affect T cell functions, while the internalization of TEXs is not required for signal delivery that causes changes in gene expression. In contrast, many authors argued that T cell functions can still be impaired by internalizing TEXs, although They are more difficult in internalizing TEXs than other immune cells. Vignard and colleagues 13 found that resting or activated CD8 + T cells can internalize melanoma-derived exosomes as early as 5 h after exposure under electron microscopy or confocal microscopy, consistent with the previous studies. 14 Moreover, the nucleic acids in the TEXs, especially mRNA and miRNAs, are also responsible for functional changes of T cells, which indicates that TEXs can reprogram the recipient T cells through internalization. 15 For example, TEXs downregulate the inhibitory genes in CD4 + T cells and result in a loss of CD69 expression on the surface of T cells. 11 When transfected into normal T cells, the RNA purified from TEXs can change the T cell function. 16 Together, TEXs can influence T cell functions through either receptor-ligand binding or internalization.

TEXS DELIVER IMMUNOSTIMULATORY SIGNALS TO T CELLS
A variety of molecules on the surface of TEXs, such as major histocompatibility complex (MHC) I and II, tumor-associated antigens (TAA), HSP70 and CD40, are thought to enhance T cell-mediated immunity against tumor through dendritic cells (DCs). [17][18][19] DCs that were stimulated by TEXs can simultaneously promote the proliferation of both CD4 + and CD8 + T cells and promote the differentiation of CD8 + T cells into CTLs, thereby enhancing the anti-tumor ability in vitro and in vivo. 20 DCs to release IL-6 to block Treg cell differentiation, which   is TGF-β-dependent, and promote Th17 cell differentiation. 24 Interestingly, IL-12-anchored exosomes can directly induce T cell proliferation and enhance their cytotoxic effect by reversing the suppressed   JAK2/STAT5 pathway. 25,26   3  TEXS DELIVER INHIBITORY SIGNALS  TO DIFFERENT T CELL SUBSETS   3.1 TEXs deliver inhibitory signals to CD8 + T cells When binding to their cognate receptors, the inhibitory ligands on the TEXs, such as TGF-β, PD-L1, CD39 and CD73, can deliver negative signals to recipient T cells. The TEXs-induced immunoinhibitory responses vary among different T cells subsets ( Figure 1). TEXs mainly inhibit CD8 + T cell activation and promote its apoptosis and exhaustion. Several mechanisms have been described to inhibit their activation. First, through TGF-β pathway TEXs suppress the response of CD8 + T cells to IL-2, a key cytokine essential for T-cell activation and proliferation. 27,28 Second, the JAK/STAT pathway is crucial for the function of cytokines sharing the γ-chain of the IL-2 receptors, such as IL-2, IL-7 and IL-15; TEXs can reduce JAK3 expression and diminish cytokine productions, thereby inhibiting activation of CD8 + T cells. [28][29][30] Third, TEXs are able to activate NF-κB signaling pathway of CD8 + T through Toll-like receptor 2/4 (TLR2/4), which leads to IL-6 upregulation and subsequent STAT3 activation. 31 37 The signaling pathway after PD-1 binding to PD-L1 may involve SHP-1/2, TCR and their downstream signaling, such as ZAP70, PI3K, PKB/AKT, mTOR, RAS, MAPK/MEK and ERK [37]. Additionally, exosome PD-L1 was significantly increased in the responders during the early stages of immunotherapy, suggested that TEX-PD-L1 is a marker of adaptive immune activation. 38 The mechanisms underlying the induction of CD8 + T cell apoptosis by TEXs have been extensively studied. 7,39,40 TEXs express FasL (Fas Ligand), a transmembrane type II protein belonging to the TNF protein superfamily, which plays a pivotal role in Fas receptor -mediated apoptosis of CD8 + T cells. [41][42][43] Priyanka et al 32 found that apoptosis of CD8 + T cells is TEXs dose-dependent, and can be neutralized by anti-Fas (ZB4) mAbs. Moreover, the FasL on the TEXs downregulates the TCR/CD3ζ expression in T cells, which is correlated with a poor F I G U R E 1 The immunosuppressive effect of TEXs on different subsets of T cells TEXs suppress different subsets of T cells through delivering the nucleic acids or inhibitory proteins such as mRNA, PD-L1, TGF-β, Galectin-9 and FasL to recipient T cells. For CD8 + T cells, TEXs inhibit its activation mainly through TGF-β and PD-L1/PD-1 signal pathways. In addition, TEXs induce CD 8 + T cells apoptosis through Fas/FasL pathway and exhaust by activation of immune checkpoint such as PD-L1/PD-1. TEXs also induce apoptosis of CD4 + T cells, inhibit their proliferation, as well as promote them to differentiate into Treg cells. On the contrary, TEXs promote Treg proliferation and inhibit apoptosis, thus enhance their immmunoinhibitory effects prognosis in several tumors. 44 TEXs can also reduce the expression of JAK3 and up-regulate the proapoptotic Bax levels of CD8+ T cells to induce apoptosis. 45 Alternatively, TEXs promote p38MAPK phosphorylation that induces endoplasmic reticulum stress, which activates the PERK-eIF2α-ATF4-CHOP signaling axis, eventually leading to CD8 + T cell apoptosis. 46 Of note, TEXs expression PD-L1also promote CD8 + T cells apoptosis through PD-1/PD-L1 interaction. 34 In addition to participation in the activation and apoptosis of CD8 + T cells, TEXs impair the effector function of CD8 + T cells, leading to T cells dysfunction, i.e., T cells exhaustion. During acute infection, when the antigens are cleared, most T cells will normally die to protect normal tissues from attack by the over-activated T cells. In cancer, T cells receive persistent stimulation of numerous antigens arising from cancer cells and TEXs, memory fails to efficiently develop and T cells become exhausted. Exhausted T cells are characterized by the high expression of inhibitory receptors, including PD-1, TGF-β, TIM-3, CTLA-4, TIGIT, LAG-3 and BTLA, and loss of effector functions such as cytokine production and ex vivo killing capacity. 47,48 Several mechanisms have been proposed for T cell exhaustion. PD-L1 is a membranebound immunoinhibitory ligand that is often overexpressed in tumor cells. Tumor cells can secrete PD-L1 through TEXs, which binds to its receptor PD-1 on the surface of recipient T cells. 49,50 Usually, the level of circulating TEX-PD-L1 positively correlated with tumor burden, and the high levels of TEX-PD-L1 reflect poor outcomes, which seem to be a more reliable biomarker than PD-L1 expression in tumor biopsy. 51 Chen et al [52] found the level of TEX-PD-L1 was significantly increased by IFN-γ treatment, and the binding of TEXs to PD-1 was increased on CD8 + T cells. TEX-PD-L1 is more easily to interact with activated CD8 + T cells, but not inactivated counterparts, and inhibited the proliferation of CD8 + T cells, production of cytokines (IFN-γ, IL-2, and TNF) and cytotoxicity (inhibit their granzyme B secretion). 35,52 Therefore, a high level of exosomal PD-L1 is associated with disease progression and poor prognosis, and neutralization of exosomal PD-L1 by anti-PD-1/PD-L1 mAb will reverse TME and induce endogenous antitumor immune response. 6 However, the PD-L1 on the TEXs is not associated with the PD-L1 expression level in cancer tissues. 53 Interestingly, even in models resistant to anti-PD-1 mAb, the removal of exosomal PD-L1 can still inhibit tumor growth and enhance systemic memory immune activity, which has become a new strategy for immunotherapy. 6 Of note, PD-L1 can transfer to both tumor cells and immune cells, such as macrophages and DCs, through TEX-PD-L1, which play an important role in the regulation of immunity in TME. 35 Interestingly, It was reported that CLL cell-derived exosomes also express PD-L1 and induce CD19-directed chimeric antigen receptor T (CART19) cell exhaustion. 54  In addition, Maybruck et al. 16 found that galectin-1 (Gal-1), an immunoregulatory protein in TEXs, is responsible for transforming normal CD8+ T cells into a suppressor phenotype (SP). Importantly, these SP phenotypes of CD8+ T cells lack CD27/CD28 expression, and suppress the function of normal T cells. 16,56,57 The inhibitory NKG2D ligand, MIC A/B, binds to NKG2D, down-regulates NKG2D expression and inhibits CD8+ T cells-mediated cytotoxicity. 58 In hepatocellular carcinoma, TEXs induce CD8+T cell exhaustion by delivering 14-3-3ζ, an immunosuppressive molecule that promotes the proliferation of cancer cells and induces epithelial-mesenchymal transition (EMT). 59 The miRNA within TEXs play an important role in T cell exhaustion. For example, Hsa-miR-498 can inhibit cytokine synthesis such as TNF-α and CD8 + T cells-mediated cytotoxicity in vivo and in vitro. 46,60 Recently, accumulating evidence suggest that TEXs can reprogram the cell metabolism to facilitate cancer progression, angiogenesis, metastasis, drug resistance and immunosuppression. 10,61 Similarly, TEXs also can modulate CD8 + T cell function through alteration of cellular metabolism. 62,63

TEXs deliver inhibitory signals to CD4 + T cells
TEXs inhibit proliferation of CD4 + T cells by impairing its response to IL-2 through TGF-β pathway and promote cell apoptosis. 64 Huang et al. 65 found that silencing TGF-β in TEXs promotes CD4 + T-cell proliferation and Th1 cytokines production. The immunoinhibitory molecules, CD39 and CD73, are expressed on the surface of TEXs, and can convert ATP into adenosine, which significantly suppresses CD4 + T cell proliferation. 12,66 Besides inhibiting cell proliferation, TEXs also induce CD4 + T cell apoptosis. Zhou et al 67  Unlike CD8 + T cells, CD4 + T cells can differentiate into Treg cells that are CD25 + and Foxp3 + , and suppress immune response. 71 TEXs can induce Treg cell differentiation through PD-L1/PD-1 signaling pathway, and block differentiation towards CD4 + IFN-γ + Th1 cells. 72 Also, TGF-β is essential for FOXP3 expression, which not only inhibits T cell proliferation but also promotes Treg cell phenotype differentiation. 65,73 Conversely, when TGF-β is neutralized with mAb or silenced by shRNA, the immunosuppressive effect of TEXs on other immune cells will be alleviated. 74 TEXs also promote the expansion of Treg cells and confers the resistance to apoptosis via TGFβ and IL-10. 75,76 Moreover, TEXs can recruit Treg cells to the tumor through CCL20, thereby inhibiting the proliferation of other T cell subsets. 69,76 TEXs also enhance the immune inhibitory function of Treg cells, which is mainly mediated by the CD73 and CD39 on the surface of TEXs. 12,77 What's more, MHC class II molecules, as ligands of LAG3, may enhance Treg cells function through TEXs. 78 Additionally, the inhibitory molecules, such as galectin-9 (TIM3 ligand), CD160(BTLA ligand), can also exert immunosuppressive effects through TEXs. 30

Indirect T cell inhibition by TEXs
Besides direct signal transfer to T cells, TEXs can regulate T cell functions through other immune cells, such as DCs, macrophages, myeloidderived suppressor and NK cells ( Figure 2).
As effective antigen present cells, DCs have dual regulatory functions in TME. DCs can extract and process TAAs from TEXs, and then present the antigens to T cells to elicit anti-tumor immune response. 7 Therefore, TEX-loaded DCs may be used as cancer vaccines to improve therapeutic response. Marton et al. 79   cell cytotoxicity is suppressed when cells are exposed to MICA *008, a human NKG2D ligand releasing from TEXs. 90 Also, TEXs can attenuate the response of NK cells to IL-2, a crucial cytokine that stimulate NK cell expansion and the release of perforin. 91 TEXs from CLL can be internalized by stromal cells, and the microRNA and proteins are delivered to transform stromal cells into inflammatory phenotypes, which secrete inhibitory cytokines and promote CLL cell survival. 92

TEXs are valuable markers for the diagnosis, prognosis, and therapeutic choices in cancer patients
In the precedent years, TEXs have attracted increasing interest in the field of liquid biopsy. As previously described, they can be isolated from almost all human biological fluids, such as blood, urine, amniotic fluid and saliva. The proteins, nucleic acids and other molecules in TEXs contain a large amount of information about tumor antigens, genetic material, and immune stimulating molecules. Therefore, TEXs are helpful in the diagnosis of disease, especially for patients with difficulty in obtaining tissue biopsy. 93 In addition, TEXs have important clinical prognostic value as biomarkers. Elevated levels of TEXs in liquid biopsies of cancer patients are usually associated with a higher tumor burden, and immunosuppressive molecules of TEXs, such as PD-L1, CTLA-4, TIM3 in the TEXs are also correlated with poor prognosis or disease progression. 53,94 Also, genetic materials such as microRNA, lncRNA, circRNA and DNA are associated with cancer progression and prognosis. [95][96][97] Exosome DNA usually contains a variety of clinically relevant tumor-specific mutations such as EGFR, BRAF, RAS, IDH, and HER2, which making it a promising therapy recommendations for "liquid biopsy". 98 Nowadays, TEXs are undergoing extensive clinical trials as a biomarker for disease diagnosis, prognosis and immunotherapy in different cancer patients, which will give us more insights in the future (  99 Kosgodage et al 100 reported that chloramidine/Bisindolylmaleimide, a kind of microvesicles release inhibitors, is effective to enhance cancer chemotherapy efficacy. In addition, the reduction of TEXs may also improve the TME, thus enhance the function of immune cells, especially T cells.

TEX-based cancer vaccines
TEXs have potential application in the development of cancer vaccines due to its ability to stimulate specific antitumor immune responses via TAA and costimulatory molecules within. 38 The uptake of TEXs by DCs can induce antigen-specific CTL responses, increase the number of CD8 + T cells, and activate CD4 + T cells, thus enhancing antitumor immunity. 101 In previous studies, TEX-carrying DC immunotherapy has been shown to improve survival. 102 However, in some types of tumors, stimulation of exosomes may lead to immune tolerance of DCs, therefore, another possibility to consider is to use exosomes of DCs that were previously stimulated by tumor cells.

CONCLUSIONS
Although having immunostimulatory effects on T cells, TEXs induce immunosuppressive effects predominantly in TME through both direct and indirect mechanisms. TEXs impair the function of CD8 + T cells by directly inhibiting the activation, proliferation and cytotoxicity, as well as promoting apoptosis. Other immune cells, such as DCs, macrophages, myeloid-derived suppressor cells and NK cells contribute to indirect immune suppression by TEXs. Of note, exosomes from CAR-T cells represent a novel strategy of cancer immunotherapy and merits further investigations.