Exosomes carrying immunoinhibitory proteins and their role in cancer

Summary

Recent emergence of exosomes as information carriers between cells has introduced us to a new previously unknown biological communication system. Multi‐directional cross‐talk mediated by exosomes carrying proteins, lipids and nucleic acids between normal cells, cells harbouring a pathogen or cancer and immune cells has been instrumental in determining outcomes of physiological as well as pathological conditions. Exosomes play a key role in the broad spectrum of human diseases. In cancer, tumour‐derived exosomes carry multiple immunoinhibitory signals, disable anti‐tumour immune effector cells and promote tumour escape from immune control. Exosomes delivering negative signals to immune cells in cancer, viral infections, autoimmune or other diseases may interfere with therapy and influence outcome. Exosomes can activate tissue cells to produce inhibitory factors and thus can suppress the host immune responses indirectly. Exosomes also promise to be non‐invasive disease biomarkers with a dual capability to provide insights into immune dysfunction as well as disease progression and outcome.

Introduction

Extracellular vesicles (EVs) are emerging as a newly recognized intercellular communication system that operates in all uni‐ and multi‐cellular organisms 1. The EVs were first identified by electron microscopy in the late 1970s, when maturing reticulocytes were observed to release 50 nm microvesicles (MVs) upon fusion of multi‐vesicular bodies (MVB) with the cellular plasma membrane 2, 3. Initially, MVs were considered as the mechanism for elimination of cellular waste, and only recently has their role as biologically significant vehicles for horizontal transfer of molecular signals and genes between cells been recognized 4. Today, the nomenclature used for these MVs has changed, due largely to the realization that cells release a variety of EVs, comprising vesicular bodies of different sizes and of different cellular origins. The largest vesicles are apoptotic bodies (1000–5000 nm). MVs are intermediate‐sized (200–1000 nm) and are shed as membranous ‘blebs’ from the cell surface. Exosomes are the smallest subset of EVs (30–150 nm) that differ from other EVs not only by size, but also by their endosomal origin and the molecular and genetic cargo which, in part, reflects that of the parent cell 5, 6. Exosomes are formed from the endosomal compartment of parent cells by intraluminal invaginations of the membrane in late endosomes 7, 8, and their cargo is enriched in proteins characteristic for endosomes such as ALG2‐interacting protein X (ALIX), tumour susceptibility gene 101 (TSG101) or syntenin‐1, attesting to their endosomal origin 6, 8. When mature MVBs filled with populations of small vesicles fuse with the parent cell surface membrane, exosomes are released into the extracellular space.

The nomenclature attempting to divide EVs into subsets based on size is imperfect; a great deal of confusion exists because of potential overlaps and emerging evidence that larger EVs may carry more and different cargo components than exosomes 9. For this reason, it is critical to explain that the term ‘exosome’ used in this paper is applied to tumour‐derived vesicles that are isolated from tumour cell supernatants or plasma of patients with cancer by size‐exclusion chromatography yielding a well‐defined fraction of 30–150 nm‐sized, morphologically intact vesicles that carry endocytic markers and a cargo derived from parent tumour cells.

Exosomes as unique delivery vesicles

As indicated above, the molecular content of exosome membrane is derived in part from the parent cell surface and in part from the endosome. In addition to tetraspanins (e.g. CD9, CD63, CD81), often used as EV markers, exosomes carry the MVB‐related proteins not present in EVs formed by ‘blebbing’ of the parent cell membrane. The exosome lumen contains components of the parent cell cytosol as well as various RNA species, and possibly some DNA 10, 11. Exosome formation and secretion are regulated strictly in the parent cell and have been examined extensively 12. While exosome secretion is a normal physiological activity, cells experiencing stress or cells in pathological conditions release masses of exosomes which are found in all body fluids. The exosome biogenesis and their molecular/genetic content, which approximates that of the parent cell, suggest that exosomes have the potential to serve as non‐invasive disease biomarkers. For this reason, exosomes are of special interest among various EVs, and their roles in diagnosis, prognosis and outcome of various human diseases are currently of great interest.

Emerging evidence suggests that exosomes participate in normal physiological activities, modulating tissue differentiation and repair, haematopoietic stem cell development, blood coagulation, pregnancy or immune surveillance 13, 14, 15, 16, 17. Exosomes also participate in various pathological processes, including tumour growth and metastasis as well as chronic inflammation and infections 18. The list of human diseases in which exosomes mediate pathology extends to HIV‐1 19, autoimmunity 20, Parkinson's disease 21, Alzheimer's disease 21, numerous inflammatory conditions 22 and others. Perhaps of special significance for the pathological role of exosomes is their effect on the host immune system. Exosomes produced by malignant or virus‐infected cells carry an immunosuppressive cargo and induce dysfunction of immune effector cells not only locally at the disease site, but also systemically. The mechanisms used by exosomes to reprogramme immune cells in a way that leads to suppression of immune responses and promotion of the disease have been studied most extensively in cancer. Tumours produce and use immunoinhibitory exosomes to reprogram immune cells and to orchestrate escape from the host immune system 23. As the immune system plays a pivotal role in human disease, the mechanistic insights into tumour‐derived exosomes (TEX) and their effects on immune cells in cancer are likely to be translatable, in part, to other pathological conditions. At the very least, they will provide a roadmap to understanding of how the exosome‐mediated communication system contributes to human disease progression and outcome.

TEX and their characteristics

Tumour cells grown in cultures are avid producers of exosomes. All exosomes are tumour‐derived in supernatants of such cultures, and most recent studies aimed at exosome characterization used TEX isolated from cell line supernatants. Upon examination by transmission electron microscopy (TEM) (Fig. 1), TEX appear to be well‐formed, spherical, membrane‐bound vesicles heterogeneous in size (often < 50 nm in diameter). The TEX membrane is decorated by numerous molecular species, including proteins, lipids and glycans, with the topography reminiscent of that found in the parent cell membrane 24, 25. The TEX lumen contains nucleic acids, microRNAs, other RNA species and possibly DNA, as well as a plethora of proteins, including various soluble factors, chemokines and numerous enzymes. Using Western blots or immune arrays, TEX have been found to carry various immunoinhibitory molecules, as shown in Fig. 1b. Immuno‐EM has been used to demonstrate the presence of such biologically significant molecules as CD95 ligand (FasL), programmed cell death ligand 1 (PD‐L1) or glypican‐1 in the TEX membrane 26, 27. TEX produced by different tumour cell lines carry distinct molecular signatures 28, 29. Proteins and RNA species that have been identified in TEX by mass spectrometry are deposited in the ExoCarta database, accessible online 30. TEX also carry tumour‐associated antigens (TAAs) expressed by the parent cell. The proteins identified most commonly in TEX are major histocompatibility complex (MHC) class I and II molecules, CD63, heat shock proteins, actin, tubulin and components of such cellular signalling pathways as phosphatidylinositol (PI3) kinase, mitogen‐activated protein kinase (MAPK), β‐catenin, Wingless‐related integration site (WNT), Notch pathways and others 31, 32. TEX also carry oncogenes or oncogenic proteins and have been dubbed as ‘oncosomes’ 33. The molecular and genetic contents of TEX are being interrogated intensively, because they are perceived as potentially useful future cancer biomarkers 27, 34. In this context, it is important to note that exosomes isolated from supernatants of cell lines established from pathological tissues carry biomarkers characteristic of a given disease such as TAAs in cancer, viral antigens in infections, amyloid‐β and tau in Alzheimer's disease or miRNAs regulating osteoblast and osteoclast functions in rheumatoid arthritis 19, 21, 35, 36.

Figure 1.

(a) Transmission electron microscope (TEM) images of tumour‐derived exosomes isolated by the mini‐SEC method (described in 28) from supernatants of PCI‐13, a human head and neck cancer (HNC) cell line. Exosomes placed on a copper grid were stained with 1% uranyl acetate in ddH₂0. (b) Western blots of exosomes isolated by the mini‐SEC method from plasma of two patients with HNC. These exosomes carry various immunoinhibitory proteins. Courtesy of Dr Sonja Funk 29. Exosomes isolated from plasma of normal donors variably carry some of the same proteins but at much lower expression levels as shown in 29.

TEX‐mediated information transfer

TEX are well equipped for the task of information transfer, and they modulate a variety of physiological and pathological activities. They do so by the delivery of information to neighbouring or distantly located recipient cells and reprogramming their functions. Tumour cells, which are normally under oxidative stress, secrete excessive numbers of TEX 37, which distribute freely throughout all body fluids. Thus, TEX effectively establish a communication network between the tumour and host cells. TEX also transfer information from one tumour cell to another and can deliver autocrine signals 38. TEX isolated from plasma of cancer patients have been shown to exert biological activities in vitro and in vivo following injections into experimental animals 39.

The mechanisms through which TEX alter functions of recipient cells are only partly understood. It appears that using one or more mechanisms of cell entry, such as receptor/ligand signalling, fusion, phagocytosis or endocytosis, TEX deliver to recipient cells the membrane‐protected content in the form specified by parent cells. Following internalization, TEX disrobe and deliver mRNA, miRNA and DNA to recipient cells. These molecules may integrate into the cell machinery to initiate recipient cell reprogramming 40, although it is likely that in addition to internalizing nucleic acids, which induce changes in the recipient cell transcriptome, exosomes also deliver signals modifying the proteome (Table 1). Perhaps the best known and most widely quoted example of the TEX ability to alter cellular functions is reprogramming of the bone marrow microenvironment by melanoma‐derived TEX 57. These exosomes, upon in‐vivo transfer to the murine bone marrow, transformed it into a prometastatic niche promoting the development of melanoma and interfering with normal haematopoiesis. Evidence from multiple recent studies confirms the ability of TEX to alter functions of various recipient cells, including immune cells 35, 45, 58. Interestingly, T lymphocytes, unlike other mononuclear cells, do not internalize TEX readily 41. Instead, TEX interacting with surface molecules present on T cells deliver signals which initiate a Ca²⁺ flux and activate downstream signalling, resulting in alterations of the recipient cell transcriptome and reprogramming of T cell functions 41. Various immune cells differ in their ability to internalize and process TEX. While T cells interact with TEX mainly via the receptor/ligand‐mediated signalling, other lymphocytes and monocytes internalize TEX rapidly 41. Possibly, recipient cells determine the mode that TEX employ for delivery of their cargo, or multiple entry mechanisms may be used either simultaneously or preferentially by TEX, depending on the molecular cargo they carry. TEX deliver signals to and reprogram not only immune cells but also non‐immune and tissue cells, including parent tumour cells via juxtacrine and autocrine interactions, respectively (Fig. 2). Immune cells in the periphery are reprogrammed through paracrine mechanisms mediated by TEX circulating freely and distributed in body fluids. The overall result of reprogramming appears to be the promotion of parent tumour cell growth to which reprogrammed cells in the tumour microenvironment (TME) and in the periphery contribute through secretion of soluble factors and cytokines (Fig. 2). TEX initiate and deliver signals that, simultaneously, can promote tumour progression and restrain immune cells from eliminating the tumour.

Table 1.

Effect of TEX on functions of immune cells

DIRECT EFFECTS
Receptor‐ligand type signalling *
Targeted cell	Function	Reference
T cells	Sustained Ca2+ flux	41
T cells	↓TCRzeta chain expression	42, 43
T cells	↓CD25 signalling	44
T cells	↓CD69 expression	45
T cells	↑↓ Transcription factors (JAK, STATs)	42
CD8+ T cells	↓PI3K/AKT activity	46
CD8+ T cells	↓ Proliferation	35, 41
CD8+ melanoma‐reactive T cells	↓ Proliferation	35
CD8+ T cells	↑ Apoptosis	28, 35, 46
CD4+ T cells	↑ Proliferation	35
NK cells	↓ NKG2D expression	47, 48
NK cells	↓ Cytotoxicity	47, 48
B cells	↑ Conversion to Breg	49
Monocytes	↑ Conversion to MDSC	50, 51
	↑Secretion of inflammatory cytokines	23
	↓HLA‐DR expression ↑arginase secretion, altered STAT‐3 signalling	52
Neutrophils	↑ Recruitment of PMNs to lung metastatic niche	53
TEX internalization †
T cells	mRNA‐mediated transcriptome changes	45
	miRNA‐mediated transcriptome changes	22, 54
INDIRECT EFFECTS
Treg	↑ Proliferation ↑suppression	55, 56
MDSC	↑ Suppressor function	50, 51
Breg	↑ Suppressor function	49

*Tumour‐derived exosomes (TEX) carry immunosuppressive ligands embedded in the membrane (see Fig. 1b) which interact with cognate receptors on the surface of immune cells. ^†Reprogramming of immune cells which internalize TEX [B cells, natural killer (NK) cells, monocytes] is mediated by the genes (mRNA, miRNA, DNA) delivered by the TEX. JAK = Janus kinase; STAT = signal transducer and activator of transcription; P13K/AKT: phosphatidylinositol‐3‐kinase/protein kinase B; NKG2D = natural killer cell lectin‐like receptor K1; MDSC = myeloid‐derived suppressor cell; B_reg = regulatory B cell; PMN = polymorphonuclear neutrophils; HLA‐DR = human leucocyte antigen D‐related.

Figure 2.

Signalling by exosomes produced by a pathological parent cell can promote pathology and restrain immune cells from eliminating the abnormal parent cell simultaneously. Exosomes can mediate autocrine signals (blue arrows) that lead to promotion of the parent cell activities (proliferation, differentiation, migration). Exosomes also signal to the neighboring tissue cells (juxtacrine signalling), bind to cognate receptors on these cells, reprogramme their functions and induce secretion of cytokines or chemokines which favour the parent cell, promoting its growth (red arrow). Exosomes also deliver signals to the nearby or distant immune cells (paracrine signalling), inhibiting their migration and ability to eliminate the abnormal cell. Exosomes also reprogram immune cells to produce soluble factors promoting growth and survival of the pathological parent cell (black arrows). Exosome signalling is driven by the parent cell and is contextual.

TEX‐mediated inhibition of immune effector cells

Delivery of TEX to immune cells has been shown to result in various levels of dysfunction of these cells 35, 42. All types of immune cells are sensitive to TEX‐mediated interference. However, T lymphocytes which seem to interact with ligand‐carrying TEX via cognate surface receptors are especially vulnerable to negative signals delivered by TEX. We have shown that TEX deliver receptor‐mediated signals to T cells that result in a sustained Ca²⁺ flux 41 and subsequent activation of the relevant downstream pathways, culminating in alterations of the recipient cell transcriptome and of responder cell functions 41, 45. To determine how TEX‐delivered signals translate into transcriptional activity and functional changes in recipient T cells, we isolated CD4⁺, CD8⁺ and CD4⁺CD39⁺ regulatory T cell (T_reg) subsets from human peripheral blood of normal donors and co‐incubated these cells with TEX 45. We measured expression levels of 24 immunoregulatory genes by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) in the cells ± TEX. Massive changes in expression levels of multiple genes were observed following co‐incubation with TEX, including changes in genes regulating immune suppression or immune activation. Multi‐factorial analysis of ΔCt values showed that the presence or absence of exosomes, the recipient cell type and the activation status of the recipient cell were the only factors that regulated TEX‐induced transcriptional activity significantly in T cells 45. The observed massive changes in mRNA expression levels were induced equally by co‐incubation with TEX or DEX [exosomes produced by human monocyte‐derived cultured dendritic cells (DC)]. However, TEX and DEX modulated different immunoregulatory genes, and some of the genes were modulated differently in T_reg than in CD4⁺ or CD8⁺ effector T cells. To show that TEX‐mediated signals translated into relevant functions, we concomitantly measured CD69 (an activation marker) expression in CD4⁺ T effector cells by flow cytometry. TEX decreased expression levels of CD69 significantly on the surface of CD4⁺ T cells, which was consistent with TEX immunosuppressive functions. Also, T_reg co‐incubated with TEX, which carry both CD39 and CD73 ectonucleotidases (Fig. 1b and 55), up‐regulated production of immunosuppressive adenosine significantly in a concentration‐ and time‐dependent manner 55. This set of data, together with the demonstration that CD8⁺ or CD4⁺ T cells did not internalize TEX, provided evidence that TEX signalling by engaging surface receptors on recipient T cells modulated T cell responses negatively.

The question may be asked which receptors on activated T cells are targeted by TEX. We have reported previously that TEX regulate functions of the T cell receptor (TCR) negatively by inducing down‐regulation in expression levels of the CD3‐associated zeta chain 42, 43. TEX appear to be responsible for low TCR/zeta expression levels seen frequently in circulating T cells of cancer patients, especially those with advanced malignancies 59. Low TCR zeta chain expression on circulating and tumour‐infiltrating T cells has been linked to immune dysfunction and poor outcome in patients with breast cancer 60. Co‐incubation of human T cells isolated from peripheral blood of healthy donors with TEX results consistently in down‐regulation of the TCR zeta chain expression levels 43. Functions of the interleukin (IL)‐2R, another key receptor in T cells, are also modulated negatively by co‐incubation with TEX 44. TEX reduce Janus kinase (JAK) expression and phosphorylation in activated T cells 35, and as the integrity of the JAK pathway is essential for functions of IL‐2, IL‐7 and IL‐15, the cytokines sharing the γ chain of the IL‐2R, down‐regulation of JAK activity by TEX is detrimental to T cell proliferation 42. In our initial experiments, TEX were shown to inhibit proliferation of activated CD8⁺ T cells significantly but to promote, albeit less convincingly, expansion of CD4⁺ T cells, while exosomes released by normal cells promoted proliferation of all T cells 35. Consistent with these data, TEX were found to increase signal transducer and activator of transcription (STAT)‐5 phosphorylation in activated CD4⁺ T cells and to inhibit STAT‐5 phosphorylation in activated CD8⁺ T cells 42. These data suggest that TEX can modulate functions of transcription factors such as JAKs and STATs in recipient T cells.

Do TEX target immune cell subsets selectively?

We next considered whether tumour‐reactive T effector cells were more sensitive than non‐tumour‐reactive T cells to TEX‐mediated suppression. In co‐incubation experiments, TEX inhibited preferentially the proliferation of human melanoma‐specific CD8⁺ T cells generated in cultures of T cells with melanoma peptide‐pulsed DC 35. Conversely, our more recent experiments show that exosomes isolated from plasma of patients with melanoma carry melanoma‐associated antigens including gp100, tyrosinase‐related protein 2 (TYR2) or melan A (MART1), as well as MHC class I molecules. This suggests that melanoma antigens carried by TEX might be recognized by CD8⁺ T cells and generate responses. However, as tumour‐specific antigens are delivered as a ‘bundle’, together with numerous suppressive molecules, generation of antigen‐specific CD8⁺ T cells is unlikely in the TME. We reported that activated CD8 ⁺ T cells enriched in CD95 or PD‐1 on the cell surface were especially sensitive to apoptosis mediated by FasL⁺ or PD‐L1⁺ TEX 28, 35. Further, TEX‐mediated signals leading to apoptosis of activated CD8⁺ T cells were associated with early membrane changes (i.e. annexin V binding) in these T cells, caspase 3 cleavage, cytochrome C release from mitochondria, loss of mitochondrial membrane potential (MMP) and DNA fragmentation 46. These data demonstrated that TEX can induce apoptosis in activated CD8⁺ T cells by engaging the extrinsic as well as intrinsic apoptotic cascades. The phosphatidylinositol 3‐kinase/protein kinase B (PI3K/AKT) pathway was shown to be the key target for TEX in activated CD8⁺ T cells: dramatic, time‐dependent AKT dephosphorylation and concomitant decreases in expression levels of BCL‐2, BCL‐xL and myeloid cell leukaemia (MCL)‐1 accompanied by an increase in levels of pro‐apoptotic Bcl‐2‐associated X protein (BAX) were observed in these cells during co‐incubation with TEX 46.

Importantly, TEX are apparently able to distinguish CD4⁺ T effector cells from CD4⁺ T_reg. Effector functions (proliferation, cytokine production) of the former were inhibited by TEX, while expansion and suppressor activity of CD4⁺ forkhead box protein 3 (FOXP3)⁺CD39⁺ T_reg were enhanced 56. This suggests that TEX are able to recognize the ‘address’ of recipient T cells, an impression supported by more recent demonstration of the uptake of PKH26‐labelled TEX by T_reg in the absence of such uptake by conventional T cells 41, 45. Also, TEX‐mediated mRNA changes were qualitatively and quantitatively different in T_reg versus conventional CD4⁺ or CD8⁺ T cells 45. While preliminary, these data suggest that TEX are able to discriminate between T cells, delivering different signals to effector versus regulatory lymphocytes. Further, T lymphocytes are not the only immune cells targeted by TEX. Activities of human natural killer (NK) cells, B cells and monocytes are also altered by co‐incubation with TEX. In NK cells, down‐regulation in expression of the activating receptors, especially NKG2D, is induced by TEX carrying MHC class I polypeptide‐related sequence A (MICA) and MHC class I polypeptide‐related sequence B (MICB) ligands 47. NK cell activation and cytotoxicity is inhibited by transforming growth factor (TGF)‐β, which is displayed prominently on TEX as TGF latency‐associated protein (TGF‐LAP), the form necessary for TGF‐β activation upon binding to integrins, e.g. α6βV, on the surface of recipient cells 47, 48. TEX, which are able to make adenosine from adenosine triphosphate (ATP) by virtue of carrying CD39 and CD73 [46] are implicated in inducing suppressive activity in activated B cells, because adenosine can convert activated B cells into regulatory B cells 49. TEX have been reported to inhibit normal differentiation of monocytes and to convert monocytes into TGF‐β‐expressing DCs, which secreted prostaglandin E₂ (PGE₂) and interfered with the generation of cytolytic T cells 50, 51. In addition, TEX skewed differentiation of myeloid precursor cells towards developing into highly suppressive myeloid‐derived suppressor cells (MDSCs). This function of TEX was dependent upon myeloid differentiation primary response gene 88 (MyD88) signalling in monocytes and the presence of TGF‐β and PGE₂ in the TEX cargo 61. Importantly, TEX can also activate recipient cells to produce inhibitory cytokines and thus influence indirectly functions of cells found in the environment of recipient cells. In aggregate, TEX emerge as biologically active vesicles capable of influencing negatively functions of different types of immune cells by mechanisms engaging one or multiple molecular pathways responsible for functional changes in recipient immune cells.

Consequences of TEX‐mediated negative signalling

Tumour‐induced immune suppression has been a major barrier to immune therapy of cancer. Even today, when check‐point inhibitors are achieving unprecedented successes in overcoming tumour‐induced immune suppression, a fraction of cancer patients fail to respond to check‐point inhibition 62. It is suspected that the reason for unresponsiveness of these patients is that other check‐points interfering with anti‐tumour immune responses exist and are fuelled by cancer. TEX carrying and delivering a cargo of inhibitory biologically active molecules to immune cells may be the primary suspect.

Human cancers develop, grow and metastasize successfully because they protect themselves actively from immune intervention by the host. Tumours use multiple mechanisms for inducing immune suppression and dysregulating anti‐tumour activities of immune cells 63. TEX carrying and delivering various inhibitory ligands to immune cells in the TME and in the periphery represent one of many mechanisms that tumours use to engineer their escape from the host immune system 23. However, the uniqueness of the TEX cargo consisting of many different inhibitory molecules, including TGF‐β1, PD‐L1, CD73 and FasL, delivered in one small package, endows TEX with the ability to activate numerous suppressive pathways simultaneously in recipient cells. TEX appear to be exceptionally efficient and effective tools for inducing suppression in immune recipient cells. Seen in this context, TEX could be considered as a special category of immune check‐points operating in cancer.

It has been observed that the extent of immune dysfunction in cancer patients, to which TEX almost certainly contribute, reflects tumour progression 63. Thus, rapidly progressing, aggressive tumours suppress the host immune anti‐tumour responses most effectively, while little immune suppression is detected in early, small or indolent cancers 64. Recent data suggest that the extent of local and systemic immune dysfunction in cancer may be useful metrics for disease outcome and response to therapy. Also, the tumour immune signature, which reflects the frequency as well as the type and activity of immune cells infiltrating the tumour, is being used increasingly frequently as a measure (i.e. the immunoscore) of prognosis and response to treatment 65. Emerging correlative data linking immune cell dysfunction to plasma levels of exosomes and their immunosuppressive cargo in cancer patients suggest that TEX have the potential for fulfilling the role of non‐invasive biomarkers for cancer diagnosis, progression and outcome 66. TEX carry numerous inhibitory ligands, and preliminary data suggest that the TEX molecular content correlates with the extent immune dysfunction in patients with cancer 67. Further, TEX appear to be implicated in down‐regulation of effects of immune therapies in cancer, including antibody‐based and cellular therapies. Thus, TEX could serve as decoys for tumour antigen‐specific antibodies 68 or as highly efficient, even deadly, inhibitors of therapeutic cells transferred to patients with advanced malignancies and masses of immunoinhibitory TEX present in body fluids 69.

Addressing the role played by TEX in immune suppression and inhibition of immune therapies in cancer is an important objective. While rapid progress is being made in streamlining methods for rapid isolation of exosomes from human body fluids, separation of TEX from non‐tumour‐derived exosomes in plasma is still to be achieved. Virtually all available information about TEX comes from studies of vesicles in supernatants of tumour cell lines. In contrast to ‘pure TEX’ in these supernatants, plasma of patients with cancer contains mixtures of exosomes derived from tumour and normal cells. Thus, plasma‐derived exosomes in patients are a heterogeneous mix of vesicles with different origins and different ratios of TEX to non‐TEX.

Immune cells are also a rich source of exosomes. Therefore, molecular signatures of exosomes isolated from plasma of cancer patients probably reflect those of immune cells as well as the tumour and other tissue cells. To understand how TEX modulate functions of immune cells and to define TEX molecular and functional signatures, methods for separation of TEX from immune cell‐ and other cell‐derived exosomes present in patients’ plasma need to be developed. To this end, we and others are experimenting with methods for capture of TEX from patients’ plasma and their separation from total plasma exosomes 70. Meanwhile, total plasma exosome fractions are being used to link the total protein content and molecular as well as genetic exosome profiles to immune dysregulation in patients with cancer. These studies appear to confirm the enrichment of exosomes bearing the immunosuppressive cargo in plasma of patients with cancer relative to normal donors 28, 29. Further, these studies confirm the correlations between the exosome immunosuppressive cargo and disease stage, activity and outcome 29. The future development of TEX as ‘liquid biopsies’, together with measures of the impact of TEX upon functions of immune cells in patients with cancer, promises to improve diagnosis and prognosis of human malignancies significantly.

Conclusions

The mechanisms governing interactions of TEX with immune cells discussed above are likely to be applicable, at least in part, to pathological scenarios other than cancer. Pathologically altered parent cells release exosomes with a unique, disease‐specific cargo. Cross‐talk of these exosomes with the host immune cells, which are involved in resolving the pathology, will tend to modulate immune responses negatively. Exosomes which carry or are enriched in inhibitory molecules deliver membrane‐bound, biologically competent ‘bundles’ of inhibitory signals to recipient immune cells. Acting via surface receptors on recipient cells, these exosome‐associated inhibitory molecules can activate several cellular inhibitory pathways simultaneously, reprogramming the immune cell functions. The initial surface contact may be followed by exosome uptake into the cytosol of a recipient cell. The subsequent delivery of nucleic acids, enzymes and soluble factors carried in the vesicle lumen to the recipient cell provides fuel for its genetic reprogramming. As a result of these interactions with negatively signalling exosomes, immune cells are restrained from eliminating the abnormal pathological cells. It can be surmised that exosomes carrying stimulatory signals would tend to counterbalance the evolving immune suppression, depending upon the environmental context.

Disclosure

The author declares no potential conflicts of interests.