PD-L1 on the surface of tumor cells binds its receptor PD-1 on effector T cells, thereby suppressing their activity. Antibody blockade of PD-L1 can activate an anti-tumor immune response leading to durable remissions in a subset of cancer patients. Here, we describe an alternative mechanism of PD-L1 activity involving its secretion in tumor-derived exosomes. Removal of exosomal PD-L1 inhibits tumor growth, even in models resistant to anti-PD-L1 antibodies. Exosomal PD-L1 from the tumor suppresses T cell activation in the draining lymph node. Systemically introduced exosomal PD-L1 rescues growth of tumors unable to secrete their own. Exposure to exosomal PD-L1-deficient tumor cells suppresses growth of wild-type tumor cells injected at a distant site, simultaneously or months later. Anti-PD-L1 antibodies work additively, not redundantly, with exosomal PD-L1 blockade to suppress tumor growth. Together, these findings show that exosomal PD-L1 represents an unexplored therapeutic target, which could overcome resistance to current antibody approaches.

While evaluating mechanisms regulating levels of PD-L1 in different tumors, we discovered that cancer cells can secrete a vast majority of their PD-L1 on exosomes rather than present PD-L1 on their cell surface. Using genetic knockouts for Rab27a and nSMase2 and exogenously introduced exosomes, we show that exosomal PD-L1 from tumor cells promote tumor growth in an immune-dependent fashion. Exosomal PD-L1 suppresses T cell function in vitro and in vivo at the site of the draining lymph node. Exosomal PD-L1 appears to be resistant to anti-PD-L1 as a prostate cancer syngeneic model that is unresponsive to such therapy, is dependent on both PD-L1 and exosomes for their growth. Remarkably, even the transient presence of cancer cells deficient in exosomal PD-L1 results in long-term, systemic immunity against the cancer. A role for exosomal PD-L1 is also seen in a syngeneic colorectal model. In this model, anti-PD-L1 acts additively, not redundantly, with the suppression of PD-L1 secretion. These findings have significant implications for immunotherapeutic approaches to cancer therapy.

EVs are heterogeneous (). A particular form of EVs is exosomes, which derive from the endocytic pathway (). As endosomes mature, vesicles bud inward and are released in the lumen forming intravesicular bodies within the late endosomes. These late endosomes are also called multivesicular bodies (MVB). MVBs can either fuse with lysosomes for degradation and recycling of contents or fuse with the plasma membrane releasing the intravesicular bodies extracellularly, which are then called exosomes. Exosomes can be differentiated from other EVs based on their size, morphology, density, marker expression, and dependency for specific enzymes for their biogenesis. Key enzymes in their biogenesis include NSMASE2 (aka SMPD3), which promotes budding of intravesicular vesicles, and RAB27A, which is involved in the fusion of the MVB to the plasma membrane (). Genetic manipulation of these enzymes provides an opportunity to dissect the role of exosomes in vivo.

Why the need and how to approach the functional diversity of extracellular vesicles.

It is generally thought that PD-L1 functions within the tumor bed, where cell-surface PD-L1 is directly interacting with PD-1 on the surface of tumor-infiltrating lymphocytes (TILs) (). However, PD-L1 also can be found on surface of extracellular vesicles (EVs). Furthermore, EV PD-L1 levels have been associated with tumor progression (). Whether extracellular PD-L1 can promote tumor progression by inducing a local and/or systemic immunosuppression is unknown.

PD-L1 is a membrane bound ligand found on the cell surface of many cell types that is upregulated in the setting of inflammation and/or a number of oncogenic lesions (). It binds the PD-1 receptor on immune T cells, leading to Sh2p-driven dephosphorylation of the T cell receptor and its co-receptor CD28, thereby suppressing antigen-driven activation of T cells (). This mechanism normally keeps inflammatory responses in check, and Pd-l1 knockout mice develop autoimmune-like diseases (). However, tumor cells can co-opt this mechanism to evade immune destruction. Therapeutic antibodies to PD-L1 and PD-1 block this interaction, which can then reactivate the anti-tumor immune response ().

Immunotherapy has revolutionized cancer therapy (). Immune checkpoint protein inhibitors, such as antibodies against PD-L1 (aka CD274) and PD-1 (aka PDCD1), have shown effectiveness against a large number of cancer types, including melanoma, non-small-cell lung cancer, and renal cancer. This response includes durable remissions in many patients who had previously failed multiple other therapeutic strategies. However, even in these cancers, only 10%–30% of patients respond to anti-PD-L1/PD-1 therapy (). In other cancers, such as prostate cancer, responses are rare (). The basis of differential therapeutic success between patients and between cancers remains largely unknown.

Next, we asked whether exogenously introduced exosomal PD-L1 could suppress the anti-tumor immune response and promote tumor growth. To address the effect on the immune response, we transplanted Rab27a null TRAMP-C2 cells in the flank of syngeneic mice followed by tail vein injections of in vitro collected exosomes from either their WT or Pd-l1 null counterparts ( Figure 7 A). Exosomes from the WT, but not Pd-l1 null, cells were able to induce a systemic immunosuppression as evidenced by nearly a 50% reduction in spleen size ( Figure 7 B). Furthermore, the WT exosomes were able to suppress the immune response in the draining lymph node of the Rab27a-deficient cells. Compared to the Pd-l1-deficient exosomes, the WT exosomes led to a reduced CD8/CD4 ratio, while having little effect on the T-reg cells ( Figures 7 C–7E). More importantly, they led to an increase in the fraction of cells expressing high levels of the exhaustion markers PD-1 and TIM3 and low levels of the activation marker Gramzyme B ( Figures 7 F–7K). These findings paralleled the findings seen in mice transplanted with WT versus Rab27a null cells (cf. Figures 4 and 7 ). To address the effect on the tumor growth, we transitioned to the MC38 model, given its more rapid growth characteristics. As with the TRAMP-C2 model, Rab27a-deficient MC38 cells were injected in the flank of syngeneic mice followed by exosome injection in the tail vein of the same mice. Consistent with the immune suppression seen in the TRAMP-C2 model, injection of exosomes collected from WT, but not Pd-l1-deficient MC38, cells promoted tumor growth and reduced survival ( Figures 7 L and 7M). These results confirm that exosomes are functioning through PD-L1 to suppress the anti-tumor immune response and thus promote tumor growth.

(L) Tumor growth over time following subcutaneous injection of 1 × 10 6 MC38 Rab27a null cells followed by tail vein exosomes derived from MC38 WT and MC38 Pd-l1 null cells grown in vitro. MC38 Rab27a null treated with WT versus Pd-l1 null exosomes, p < 0.01 (two-way ANOVA test).

(C–E) Flow cytometric quantification of the percentage of CD45+ CD3+ cells that are CD8 (C), CD4 (D), and regulatory T cells (T-reg) (E), respectively, in the draining lymph node (DLN).

(A) Schematic of experimental design (B)–(K); briefly, mice were transplanted with 1 × 10 6 Rab27a null TRAMP-C2 cells, followed by 3 times per week tail-vein injections of exosomes that were collected from either WT or Pd-l1 null TRAMP-C2 cells grown in vitro. Immune analysis was performed at 14 days.

Exosomes can enter the blood and lymphatic systems and travel throughout the body, potentially influencing tumor growth at distant sites (). Similarly, immune cells educated at one tumor site can travel throughout the body potentially influencing growth of tumors at distant sites (). Therefore, we next asked whether the simultaneous injection of WT and mutant cells at different sites would affect growth of either tumor. In particular, could exosomal PD-L1 released from WT tumor cells promote growth of mutant cells at a distant site, and/or could the T cells activated by the mutant cells suppress growth of WT cells at a distant site? To address this question, WT TRAMP-C2 cells were injected in one flank of each mouse simultaneously with Pd-l1, Rab27a, or nSMase2 null TRAMP-C2 cells on the other flank ( Figure 6 A). Tumor growth on each flank was then followed over time. Remarkably, growth of the WT tumor in these double injections was dramatically reduced relative to mice where WT cells alone were injected ( Figure 6 B). The effects of Pd-l1, Rab27a, or nSMase2 null cells on the distant WT tumor were similar. In contrast, the WT cells did not have any effect on the growth of Rab27a or nSMase2 null cells ( Figure 6 C). However, the WT cells did promote the growth of Pd-l1 null cells. This effect was small as tumor growth was much slower than the WT counterparts ( Figure 6 C, compare y axis to 6 B). The reason for the difference between the Pd-l1 null cells and the exosome-deficient cells is unclear and requires further study. Overall, the double-injected mice had a greatly extended survival relative to those that received WT TRAMP-C2 cells alone ( Figure 6 D). These findings strongly supported the notion that immune cells activated in the draining lymph node of the mutant side were able to travel and attack the WT tumor cells on the opposite flank. Indeed, histological analysis of the WT tumors showed a dramatic increase in the number of infiltrating lymphocytes when co-injected with the mutant cells on the opposite flank ( Figures 6 E and S7 E). Together, these data show communication between the tumors, with the effect of the mutant tumor being dominant over that of the WT tumor.

(E) Histological analysis of lymphocyte infiltration of tumors under the noted conditions. Each symbol represents an individual mouse. Representative images for each state (severe, moderate, mild, and none) can be found in Figure S7 E.

(C) Tumor growth of Pd-l1 null or Rab27a null TRAMP-C2 cells in mice singly injected or co-injected with WT TRAMP-C2 cells. n = 5 for each condition. Error bars represent SEM.

(B) Tumor growth of WT TRAMP-C2 cells in mice singly injected or co-injected with either Pd-l1 null or Rab27a null cells. n = 5 for each condition. Tramp WT versus TRAMP-C2 WT co-injected with TRAMP-C2 Rab27a null, p < 0.001. Tramp WT versus TRAMP-C2 WT co-injected with TRAMP-C2 Pd-l1 null, p < 0.001. Tramp WT versus TRAMP-C2 WT co-injected with TRAMP-C2 nSMase2 null, p < 0.01 (two-way ANOVA test). Error bars represent SEM.

(A) Schematic of the experiment: briefly, immunocompetent B6 mice were co-injected with 1 × 10 6 mutant TRAMP-C2 cells in one flank and with 1 × 10 6 WT TRAMP-C2 cells in the other flank.

The difference in the effect of Pd-l1 versus Rab27a loss on tumor growth also implied that there is an exosome independent pool of PD-L1 that is also functioning to suppress the immune response in this model. We hypothesized that unlike exosomal PD-L1, this pool may be sensitive to anti-PD-L1 antibody. This pool could explain why unlike the TRAMP-C2 model, the MC38 model is partially responsive to anti-PD-L1 antibodies (). To test this hypothesis, we evaluated the effect on survival of anti-PD-L1 alone and in combination with exosome depletion. Similar to the loss of Rab27a, the treatment of the mice with anti-PD-L1 antibody extended survival, but not to the same degree as loss of Pd-l1 ( Figure 5 G). Remarkably, the combination of Rab27a deletion and treatment with anti-PD-L1 antibodies lead to similar survival curve as the Pd-l1 deletion. This finding strongly supports the conclusion that while exosomal PD-L1 is resistant to anti-PD-L1, there is another pool in this model, likely cell-surface PD-L1, which is both a significant suppressor of the anti-tumor immune response and sensitive to the antibodies.

The difference in the effect of Pd-l1 versus Rab27a loss allowed for an epistasis analysis to address the question of whether exosomes are acting specifically through PD-L1 to influence tumor growth. If exosomes have a PD-L1 independent role, their removal should further reduce tumor growth and extend lifespan in the Pd-l1 null background. Therefore, we made Rab27a; Pd-l1 double knockout MC38 cells ( Figure S7 ). These cells grew at a rate similar to that of the Pd-l1 single mutant cells ( Figures 5 E and 5F). Thus, we conclude that exosomes are functioning predominantly, if not entirely, through their presentation of PD-L1.

Next, we asked whether the effect of exosomal PD-L1 on tumor progression is unique to the TRAMP-C2 model. To address this question, we evaluated a colorectal cancer model, MC38. Unlike the TRAMP-C2 model, this model shows a partial response to anti-PD-L1 and therapy (). Western analysis showed that MC38 cells, like TRAMP-C2 cells, secrete PD-L1 ( Figure 5 A). They also express low levels on the cell surface ( Figure 5 B). Using identical guide RNAs as in the TRAMP-C2 model, Pd-l1 and Rab27a were knocked out in MC38 cells via CRISPR/Cas9-mediated mutagenesis ( Figures 5 B and S7 A). The Rab27a knockout cells showed a loss of PD-L1 in the secreted fraction, confirming the packaging of PD-L1 in exosomes ( Figure 5 A). Also, similar to TRAMP-C2 cells, the Rab27a and Pd-l1 knockout MC38 cells showed no change in proliferation ( Figures 5 C and 5D). These cells were injected in syngeneic WT mice ( Figures 5 E and 5F). WT MC38 tumors grew rapidly, and mice had to be euthanized starting at 9 days. Loss of either Rab27a or Pd-l1 slowed tumor growth and extended lifespan. However, unlike the TRAMP-C2 model, Pd-l1 loss had a greater effect than Rab27a loss ( Figure 5 F). Therefore, exosomal PD-L1 appears to play an important, but partial role, in this model.

(G) Survival curve for mice injected with WT, Rab27a null, or Pd-l1 null MC38 cells followed by treatment with either anti-PD-L1 or isotype control antibody. n = 5 for each condition. MC38 WT isotype versus MC38 WT anti-PD-L1, p < 0.01. MC38 Rab27a isotype versus MC38 Rab27a anti-PD-L1, p < 0.05. MC38 Pd-l1 isotype versus MC38 Rab27a anti-PD-L1, ns. (log rank test).

We hypothesized that if exosomes are acting through PD-L1 to suppress the T cell activation in the lymph node, then in the absence of exosomal PD-L1, immune-competent mice will not only suppress immediate growth of the mutant tumor cells but will also develop memory toward the tumor cells. To test this hypothesis, mice injected with either Rab27a or Pd-l1 null TRAMP-C2 cells on one flank were rechallenged 90 days later with WT TRAMP-C2 cells on the other flank ( Figure 4 N). Age matched mice that had not been previously injected with any tumor cells were used as control. Remarkably, WT TRAMP-C2 tumors failed to grow in any of the mice previously injected with either mutant cell line, but showed normal growth in the age matched controls ( Figure 4 O). Thus, exposure to tumor cells lacking exosomes or PD-L1 results in a robust memory response even against cells that secrete exosomal PD-L1. We interpret this to mean that once the T cells have been exposed to tumor antigens in the absence of exosomal PD-L1, they become resistant to the suppressive effects of exosomal PD-L1.

Next, we asked whether loss of exosomes and PD-L1 have a similar effect on the immune response to the tumor. To address this question, we measured the response in the lymphoid tissues following the injection of WT, Rab27a null, and Pd-l1 null TRAMP-C2 cells ( Figure 4 A). At 14 days, the spleens of mice injected with either mutant cell line were significantly larger than those injected with WT cells ( Figure 4 B). Immunophenotyping of the spleen showed equal percentages of CD8, CD4, and regulatory T cells across the three genotypes ( Figure S6 ). These data are consistent with an enhanced generalized systemic immune response in the absence of exosomes or PD-L1. In contrast, immunophenotyping of the draining lymph nodes showed striking differences between the WT and mutant animals. CD8 positive cells made up a much greater fraction of the T cells following injection of the two mutant tumor cell lines relative to the WT line ( Figure 4 C). The fraction of CD4 was relatively down in the mutants, whereas FoxP3+ T regulatory cells composed a constant fraction of the T cells ( Figures 4 D and 4E). Given the relative increase in CD8 cells in the absence of exosomes or PD-L1, we evaluated markers of T cell exhaustion and activation within the CD8 and CD4 T cell populations. The fraction of CD8 and CD4 cells that were PD-1 high were trending down in mice receiving the mutant cells ( Figures 4 F and 4G). Much more significant was a decrease in the percentage of cells expressing the exhaustion marker Tim3 and increase in the percentage of cells expressing the activation marker Granzyme B ( Figures 4 H–4K). Furthermore, the fraction of cells positive for the proliferation marker Ki67 was significantly up among the CD8 T cells and trending up among the CD4 T cells ( Figure 4 L and 4M). These data are consistent with tumor-derived exosomal PD-L1 traveling to the draining lymph node and suppressing T cell activation.

(A–C) Flow analysis of indicated subsets of CD8+ (A), CD4+ (B) and regulatory (T-reg) (C) T cells in spleen among CD45+ CD3+ cells (n = 5 mice/genotype) 14 days post-tumor injection (1 ∗ 10 6 WT, Pd-l1 null and Rab27a null TRAMP-C2 cells). Error bars = SEM. N.S., not significant.

(O) Tumor growth volume over time following secondary subcutaneous injection of 1 × 10 6 WT TRAMP-C2 cells into immunocompetent B6 mice previously sham treated or injected with Pd-l1, Rab27a, or nSMase2 null TRAMP-C2 cells. n = 5 for each condition. Tramp WT versus TRAMP-C2 WT pre-injected with TRAMP-C2 Rab27a null, p < 0.001. Tramp WT versus TRAMP-C2 WT pre-injected with TRAMP-C2 Pd-l1 null, p < 0.001. Tramp WT versus TRAMP-C2 WT pre-injected with TRAMP-C2 nSMase2 null, p < 0.001 (two-way ANOVA test). Error bars represent SEM.

(C–E) Flow cytometric quantification of the percentage of CD8+ (C), CD4+ (D), and regulatory T cells (T-reg) (E), respectively, among CD45+, CD3+ cells in the draining lymph node (DLN) (n = 5 mice/genotype) 14 days post-tumor cell injection (1 × 10 6 WT, Pd-l1 null, and Rab27a null TRAMP-C2 cells).

It remained plausible that the exosome biogenesis factors were acting cell autonomously to suppress tumor growth, rather than non-autonomously via exosomal PD-L1 to suppress tumor growth via the anti-tumor immune response. To differentiate these alternative possibilities, we next asked whether the effect of Rab27a and/or nSMase2 loss was dependent on an active immune system. WT, Rab27a, nSMase2, and Pd-l1 null TRAMP-C2 cells were injected into NOD-scid IL2rγ(NSG)-immunodeficient mice. In striking contrast to the results seen in immunocompetent mice, the four lines led to end-stage tumors at a similar rate in the immunodeficient background ( Figure 3 E). Therefore, RAB27A and NSMASE2, like PD-L1, promote tumor growth through the suppression of the immune system.

To further confirm that the loss of Rab27a was blocking tumor growth through its role in exosome biogenesis, we repeated the experiments using nSMase2 null cells. nSMase2 was deleted again using CRISPR/Cas9-mediated mutagenesis ( Figure S5 E). Similar to PC3, nSMase2 null TRAMP-C2 cells showed a decrease in both cellular and extracellular levels of PD-L1 ( Figures S5 F–S5H). These cells were injected in syngeneic mice and compared to their WT counterparts. Similar to Rab27a null TRAMP-C2 cells, the nSMase2 null TRAMP-C2 cells failed to form palpable tumors during the time period when mice receiving WT cells had to be euthanized ( Figure 3 D). Again, a majority of the mice (8 of 10) were still alive after 90 days ( Figure S3 E). Together, these data show that blocking exosome biogenesis or removing PD-L1 result in a very similar tumor growth suppression phenotype.

Given its ability to suppress T cell activation in vitro, we wanted to know whether exosomal PD-L1 can function in vivo to promote tumor progression. To do so, we turned to a syngeneic model of prostate cancer, the TRAMP-C2 model (). This preclinical model, like human prostate cancer, is resistant to anti-PD-L1 blockade (). CRISPR/Cas9-mediated deletion of Rab27a ( Figure S5 A) and Pd-l1 ( Figure S5 B) resulted in a loss of PD-L1 in the EV fraction ( Figure 3 A). Loss of Rab27a did not influence cell-surface PD-L1 levels, nor did loss of Pd-l1 influence exosome production ( Figures S5 C and S5D). Like with the PC3 cells, deletion of Rab27a or Pd-l1 did not affect the proliferation of the cells ( Figures 3 B and 3C). The WT, Rab27a null, and Pd-l1 null TRAMP-C2 cells were injected into the flanks of C57BL6/J syngeneic mice and were followed for over 4 months. All mice injected with the WT TRAMP-C2 cells had visible tumors by around 35 days and had to be euthanized between 40 and 71 days ( Figures 3 D and 3E). In contrast, all mice injected with Rab27a null or Pd-l1 null TRAMP-C2 cells showed no tumor growth during the same time period ( Figure 3 D). Similarly, the mice injected with Rab27a and Pd-l1 null TRAMP-C2 cells showed a dramatically extended lifespan relative to their WT counterparts ( Figure 3 E). Indeed, a majority of the mice remained alive after 90 days, at which point they were used for the memory experiments described below.

Next, we asked whether exosomal PD-L1 could function similarly to cell-surface PD-L1 in the suppression of T cell activation. PD-L1 function can be measured in vitro in the setting of Raji B cell presentation of antigen to Jurkat T cells (). Normally, this presentation would activate T cells, which can be measured by secretion of interleukin-2 (IL-2). However, if PD-L1 is exogenously expressed in the Raji B cells and PD-1 is exogenously expressed in the Jurkat T cells, T cell activation is suppressed (). In this setting, we asked whether exosomal PD-L1 from PC3 cells could replace exogenous expression of PD-L1 on the Raji B cells ( Figure 2 G). As expected, in the absence of exogenous expression of PD-L1 in Raji B cells, IL-2 secretion was high ( Figure 2 H, compare first two bars). However, the introduction of the PC3 100k g extracellular fraction re-repressed IL-2 secretion showing that PC3 vesicles can replace Raji cell PD-L1 expression ( Figure 2 H, third bar). To determine whether exosomal PD-L1 was responsible for this effect, we used CRISPR/Cas9 editing to delete the Pd-l1 gene in the PC3 cells ( Figure S4 F). Deletion of the Pd-l1 gene did not affect the proliferation of the PC3 cells or alter the number of vesicles released ( Figures S4 G and S4H). However, introduction of the Pd-l1 null PC3 100k g extracellular fraction failed to repress IL2 secretion ( Figure 2 H, fourth bar). These data show that exosomal PD-L1 does function to suppress T cell activation in vitro.

Given that PD-L1 is a trans-membrane protein, exosomal PD-L1 must arise from the limiting membrane of the late endosome. The limiting membrane of the late endosome initially arises from the endocytosis of the plasma membrane. However, material provided directly from the ER and Golgi as well as cytoplasm is added as the resultant endosome matures (). Flow cytometry and immunofluorescence showed the presence of PD-L1 on both the surface and within vesicle-like structures inside PC3 cells ( Figures S4 A and S4B). Using standard cell fractionation techniques, PD-L1 and CD63 co-localized in the sucrose light, or endolysosomal fraction () ( Figure S4 C). To determine whether exosomal PD-L1 directly arises from the ER or early Golgi, we took advantage of the fact that PD-L1 is glycosylated (). This glycosylation was easily removed with the amidase PNGaseF, consistent with it being a N-linked oligosaccharide chain ( Figure S4 D). In contrast, the majority of cellular and all the exosomal PD-L1 was resistant to EndoH cleavage, consistent with maturation of the oligosaccharide chain in the distal Golgi (). Therefore, exosomal PD-L1 does not appear to come directly from the ER or early Golgi. To measure the plasma membrane as a source, we performed a cell-surface biotinylation assay ( Figure S4 E). Biotin-labeled PD-L1 was found in the cells and exosomes of treated cells, showing that exosomal PD-L1 originates from the surface of the PC3 cells.

(E) Cell surface biotinylatin assay. Left: schematic of assay. Right: Western for PD-L1 following strepavidin pull down from cells and exosomes, 48 hours following cell surface labeling. Control, not biotinylated PC3 cells.

(D) Western following treatment with different endoglycosidases. PNGaseF removes all N-linked glycosylation. In contrast, EndoH is blocked by side chain modifications occurring in the Golgi.

(C) Western for PD-L1 and CD63 following cellular fractionation based on size and density. PD-L1 is enriched in endolysomal fraction, marked by high levels of CD63.

Next, to determine if PD-L1 is specifically found in exosomes, we took a genetic approach to remove exosomes. We focused on the Rab27a and nSMase2 genes using CRISPR/Cas9-mediated mutagenesis to knock them out in PC3 cells ( Figures S3 A–S3C). Deletion of these two genes did not affect the proliferation of PC3 cells ( Figures S3 D and S3E). To follow exosomes, we ectopically expressed CD63-GFP in the three genetic backgrounds. Measurement of the conditioned media showed an almost complete absence of CD63-GFP+ particles arising from the two mutant lines versus wild-type (WT) ( Figure 2 A). To further evaluate the effect of the Rab27a and nSMase2 deletion on exosomes, we performed electron microscopy on sucrose gradient concentrated particles. The resultant images showed very few exosome-like particles in the Rab27a and nSMase2 null samples, with a greater loss in the Rab27a null cells ( Figures 2 B–2dD). Consistent with these images, western analysis showed an absence of endogenous CD63 in the 100k g preps from Rab27a null cells and a small amount remaining in the nSMase2 null cells ( Figure 2 E). In contrast to CD63 levels, nSMase2 showed a complete absence, while Rab27a null cells showed a dramatic reduction in PD-L1 in the 100k g fraction ( Figure 2 E). This difference appears to be due to an additional role for NSMASE2 on Pd-l1 transcription ( Figures S3 F and S3G). Together, these data show critical roles for both Rab27a and nSMase2 in exosome biogenesis and PD-L1 secretion.

(H) Quantification of IL-2 released by Jurkat cells following Raji presentation of super-antigen. Jurkat (PD1), Jurkat T cells overexpressing PD1; Raji (Parental), Raji B cells expressing low levels of PD-L1; Raji (PD-L1), Raji B cells overexpressing PD-L1; PC3 vesicles, 100k g pellet from conditioned media of WT PC3 cells; PC3 Pd-l1 −/− Vesc, 100k g pellet from conditioned media of Pd-l1 null PC3 cells. n = 4.

(A) Nanoparticle tracking of size and quantity of GFP + vesicles from WT and Rab27a null PC3 cells expressing CD63-GFP. Left: Density trace. Right: Integration under curve for total particles. n = 3.

Extracellular vesicles come in multiple forms differing in size, density, protein markers, and biogenesis (). Given that PD-L1 is endocytosed from the surface of cells (), we hypothesized that PD-L1 is being specifically secreted in the form of exosomes. Exosomes can be enriched relative to other vesicles based on their density by spinning the crude 100k g pellet on a sucrose gradient (). The exosomal marker CD63 traveled in the 20%–40% sucrose fractions ( Figure 1 E). PD-L1 and the additional exosomal marker HRS colocalized with CD63 ( Figure 1 F). These data support that PD-L1 is packaged in exosomes.

In vivo, tumor cells can mediate adaptive resistance by upregulating PD-L1 in response to interferon-gamma (IFNγ) released by the cytotoxic T lymphocytes within the tumor bed (). Therefore, we asked whether, in addition to upregulating cellular PD-L1, IFNγ may increase the secretion of PD-L1. Treatment of cancer cells with IFNγ led to an increase in PD-L1 in both the cellular and 100k g fractions ( Figure S2 A). The increase was proportionally similar between the two fractions, suggesting no direct impact of IFNγ on PD-L1 secretion ( Figure S2 B). In addition, IFNγ did not increase the number of vesicles secreted ( Figure S2 C). Thus, similar to the cell-surface membrane PD-L1, extracellular vesicular PD-L1 increases in response to IFNγ.

Next, we considered the possibility that PD-L1 may be differentially secreted from cells in the form of membrane vesicles. Extracellular vesicles can be enriched using sequential centrifugation transferring supernatant through increasing gravitational forces to remove cellular debris and apoptotic bodies, before finally pelleting at 100,000 gravitational forces (100k g) (). Western analysis showed 2- to 3-fold more PD-L1 in the 100k g fraction of PC3 cells relative to SK-MEL-28 cells ( Figure 1 C). This difference could have been due to more PD-L1 being loaded per vesicle or release of more vesicles. A nanoparticle tracking instrument can track the size and number of vesicles by using light diffraction off particles moving under Brownian motion (). Analysis of conditioned media from the two cell types showed a slightly elevated total vesicle count in SK-MEL-28 ( Figure 1 D). Thus, even though PC3 and SK-MEL-28 cells had similar levels of cellular PD-L1 protein, PC3 cells packaged greater amounts of PD-L1 into extracellular vesicles. This difference appears to underlie the discordance between mRNA and protein levels between the two cell lines.

Next, we evaluated potential differences in protein degradation. The two main pathways for protein degradation are the lysosome and the proteasome (). The small molecule Bafilomycin A1 (BafA1) inhibits lysosomal activity by blocking the V-ATPase hydrogen pump and thus acidification of the lysosome (). An increase in the protein LC3B, a known target of the lysosome, confirmed effectiveness of the small molecule on PC3 and SK-MEL-28 cells. However, levels of PD-L1 in both lines were unaltered, implying little turnover of PD-L1 by the lysosome in these cells ( Figures S1 C and S1D). The small molecule MG132 suppresses proteasome activity by blocking the 26S proteasome complex and, consequentially, proteolysis (). The proteasome recognizes ubiquitin side chains on its targets (). An increase in the presence of ubiquitinated proteins confirmed the effectiveness of MG132 on the two cells lines. Again though, PD-L1 protein levels were minimally affected, and, if anything, the difference of the two lines was opposite from expected as PD-L1 levels were slightly up in SK-MEL-28, but not in PC3, cells ( Figures S2 E and S2F). These results show that differences in protein stability do not explain the discordance in mRNA and protein levels.

(B) Quantification of PD-L1 in westerns. PD-L1 is normalized to GAPDH for cells and to cell number for exosomes. n = 3.

It has been reported that surface PD-L1 levels are low in prostate cancer cell lines and primary prostate tumor tissue, potentially explaining the general lack of therapeutic response to anti-PD-L1 blockade (). We sought to determine whether a transcriptional, post-transcriptional, translational, or post-translational mechanism underlies the reduced levels by comparing prostate cancer cell lines (PC3, DU145, LNCaP) to a melanoma cell line (SK-MEL-28). Measurements of mRNA and protein levels showed discordance between mRNA and protein levels across the different cancer cell lines. qRT-PCR showed a 15-fold increase of Pd-l1 mRNA levels in PC3 and DU145 relative to SK-MEL-28; LNCaP showed a near absence of transcripts ( Figure 1 A). In contrast to mRNA levels, western analysis showed similar cellular PD-L1 protein levels in PC3 and DU145 cells as SK-MEL-28 ( Figure 1 B); protein was undetectable in LNCaP cells. We asked whether the discordance in mRNA and protein levels between PC3 and SK-MEL-28 could be explained by differences in protein translation. Translation rates were determined by polysome profiling, a method by which transcripts bound by many ribosomes reflective of a high translation rate are separated from transcripts bound by one or a few ribosomes reflective of a low translation rate (). The two populations were separated on a sucrose gradient, and then the bound mRNA was measured by qRT-PCR ( Figure S1 A). This analysis showed an equal distribution of the Pd-l1 RNA across the fractions in PC3 and SK-MEL-28 ( Figure S1 B). Thus, differences in translation rates cannot explain the discordance between mRNA and protein levels between the lines.

(C) Representative western for PD-L1 in PC3 and SK-MEL-28 cells treated with or without the lysosome inhibitor Bafilomycin A1 (BafA). LC3B, positive control. GAPDH, loading control.

Differences in Translation Rates nor Protein Degradation Can Explain Discordance of mRNA and Protein Levels between PC3 and SK-MEL-28 Cells, Related to Figure 1

(E) Schematic of steps involved in exosome purification including sequential centrifugations of supernatant at increasing gravitation force and then 100k g pellet run on sucrose gradient. Location of exosomes is followed by a western blot for CD63.

(D) Nanoparticle tracking of size and quantity of secreted vesicles produced by PC3 and SK-MEL-28 cells. Left: Density plot for size. Right: Total vesicle number based on integration under the curve. n = 9.

(C) Top left: Schematic for crude isolation of secreted vesicles by sequential centrifugation of media through increasing centrifugal forces and finally pelleted at 100k g. Bottom left: Representative western blot of PD-L1 in cells versus secreted vesicles of either PC3 or SK-MEL-28 cells. Right: Quantification. n = 6.

(B) Western analysis of cellular levels of PD-L1 in the same cells as (A). Top: Representative western blot. Bottom: Quantification of three independent western blots.

Discussion

All together, these data uncover a key role for exosomal PD-L1 in enabling cancer cells to evade anti-tumor immunity. Indeed, in the presence of exosomal PD-L1, T cells in the tumor’s draining lymph node express markers of exhaustion and the spleens are smaller. Genetically blocking exosome biogenesis or deleting Pd-l1 reverses the phenotype by strongly promoting T cells activation, proliferation, and killing potential. This effect is reversed again with the introduction of exogenous exosomal PD-L1. Therefore, tumor exosomes have the ability to travel to the draining lymph node, where they present PD-L1 inhibiting T cell activation. Remarkably, blocking the release of exosomal PD-L1 not only suppresses growth of the local tumor cells but also blocks WT tumor cells injected at a distant site either simultaneously or months later. Therefore, enabling T cell activation at the local lymph node leads to a durable systemic immune response that is no longer affected by the secretion of exosomal PD-L1. The end result is extended survival of the afflicted mice.

Here, we present extensive evidence that EVs, specifically exosomes, can function to promote tumor progression by presenting PD-L1. In vitro, exosomes suppressed T cells in a PD-L1-dependent fashion. In vivo, the removal of tumor exosomes from TRAMP-C2 cells using two independent genetic mutations recapitulated the effects of deleting Pd-l1. These recapitulated effects included suppression of tumor growth, increased cellularity of the spleen, and the activation of a T cell response in lymph nodes with similar effects on the various activation, exhaustion, and proliferation markers. All these outcomes were reversed with the injection of in vitro collected exosomes carrying PD-L1. In the absence of PD-L1, the exogenously introduced exosomes had little effect. Therefore, at least in the TRAMP-C2 model, presentation of PD-L1 appears to be the major mechanism by which exosomes promote cancer progression.

This role for exosomal PD-L1 is not limited to the TRAMP-C2 model. Removal of exosomes in the colorectal MC38 model also suppressed tumor growth and extended survival. Once again, the effect was dependent on PD-L1 as the removal of exosomes had no additional effect in the Pd-l1 null background. Interestingly though, unlike the TRAMP-C2 model, the loss of exosomes alone did not have as much of an impact as Pd-l1 loss, suggesting a combined role of exosomal and cellular PD-L1 in the MC38 model. Remarkably, combining exosome loss with anti-PD-L1 treatment extended survival of these mice to a similar degree as removing PD-L1 altogether. These data show that in the MC38 model, both exosomal and cellular PD-L1 play an important role in promoting tumor progression in the latter, but not in the former, being sensitive to anti-PD-L1 therapy. Understanding what regulates the relative surface versus exosomal presentation of PD-L1 will be an important avenue of research going forward.

Chen et al., 2018 Chen G.

Huang A.C.

Zhang W.

Zhang G.

Wu M.

Xu W.

Yu Z.

Yang J.

Wang B.

Sun H.

et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Theodoraki et al., 2018 Theodoraki M.N.

Yerneni S.S.

Hoffmann T.K.

Gooding W.E.

Whiteside T.L. Clinical significance of PD-L1+ exosomes in plasma of head and neck cancer patients. Chen et al., 2018 Chen G.

Huang A.C.

Zhang W.

Zhang G.

Wu M.

Xu W.

Yu Z.

Yang J.

Wang B.

Sun H.

et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Several recent papers have identified exosomal PD-L1 in the blood of patients with a variety of cancers including head and neck cancer and melanoma (). In melanoma, it has been further shown that patients resistant anti-PD-1 therapy have elevated levels of exosomal PD-L1 in their blood prior to treatment (). Therefore, our findings on the immunosuppressive and tumor-promoting roles of exosomal PD-L1 are likely to be relevant across many cancer types. However, the degree to which exosomal versus surface PD-L1 is driving immunosuppression will vary between patients and cancer types. Identifying patients who are more or less dependent on exosomal PD-L1 will be critical going forward in deciding those who are more likely to respond to therapy. Measuring levels in the blood as shown in these recent studies is likely to be one such strategy.

Yu et al., 2012 Yu P.

Steel J.C.

Zhang M.

Morris J.C.

Waitz R.

Fasso M.

Allison J.P.

Waldmann T.A. Simultaneous inhibition of two regulatory T-cell subsets enhanced interleukin-15 efficacy in a prostate tumor model. Deng et al., 2014 Deng L.

Liang H.

Burnette B.

Beckett M.

Darga T.

Weichselbaum R.R.

Fu Y.X. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. Going forward, targeting both cell-surface and exosome presentation of PD-L1 should be considered in any therapeutic strategy. The TRAMP-C2 model is resistant to current anti-PD-L1 antibody blockade (). However, the deletion of Pd-l1 in the tumor cells had a striking effect. Similarly, although the MC38 model shows partial responsiveness to anti-PD-L1 therapy (), deletion of the Pd-l1 gene has a greater effect ( Figure 5 ). These data are all consistent with exosomal PD-L1 being resistant to current anti-PD-L1 therapy. The reason exosomal PD-L1 is resistant is unclear. It is possible that how PD-L1 is presented on the exosome makes it less responsive to the current antibodies. Alternatively, exosomal PD-L1 may be produced at high enough levels that it can compete with the delivered antibody. It is also possible that exosomes can reach targets that are sequestered from the effects of the antibody. It will be important to tease apart the interactions of exosomal PD-L1 with current therapeutics.

Ngwa et al., 2018 Ngwa W.

Irabor O.C.

Schoenfeld J.D.

Hesser J.

Demaria S.

Formenti S.C. Using immunotherapy to boost the abscopal effect. Ngwa et al., 2018 Ngwa W.

Irabor O.C.

Schoenfeld J.D.

Hesser J.

Demaria S.

Formenti S.C. Using immunotherapy to boost the abscopal effect. Potentially the most promising therapeutic implication of our findings is that inhibition of exosome secretion at one tumor site can lead to a systemic and durable immune response against distant tumor sites or secondary tumor challenges. This observation is reminiscent of the abscopal effect, originally seen in patients treated with irradiation (). In particular, irradiation of the primary tumor can lead to secondary regression of metastases. It is thought that this phenomenon is driven by activation of anti-tumor immune response and preclinical studies combining irradiation with immunosuppression are underway (). It will be interesting to determine whether localized anti-exosomal therapy combined with systemic anti-PD-L1 blockade could synergize to induce a systemic immune response against multiple tumor sites simultaneously.

Johnson et al., 2016 Johnson J.L.

Ramadass M.

He J.

Brown S.J.

Zhang J.

Abgaryan L.

Biris N.

Gavathiotis E.

Rosen H.

Catz S.D. Identification of neutrophil exocytosis inhibitors (Nexinhibs), small molecule inhibitors of neutrophil exocytosis and inflammation: druggability of the small GTPase Rab27a. Luberto et al., 2002 Luberto C.

Hassler D.F.

Signorelli P.

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Smith G.K. Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase. Phuyal et al., 2014 Phuyal S.

Hessvik N.P.

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Sandvig K.

Llorente A. Regulation of exosome release by glycosphingolipids and flotillins. Here, we used genetics to dissect the role of exosomal PD-L1 in tumor progression by deleting two important exosomal biogenesis genes: Rab27a and nSMNase2. The deletion of Rab27a led to loss of all exosomes as measured by markers (CD63, HRS), particle tracking, and electron microscopy. The deletion of nSMNase2 led to a loss of a majority, but not all, exosomes, as measured by the same assays. Therefore, both enzymes represent potential therapeutic targets. Small molecule inhibitors toward NSMNASE2 and RAB27A already exist, GW4869 and Nexinhib-20, respectively (). However, these small molecules are unable to inhibit exosome release in multiple cancer cell lines, including PC3, or show an in vivo effect in the MC38 model () ( Figures S7 D–S7H), even though their respective knockouts had a profound effect (this paper). Therefore, there is a need for more effective small molecules toward these targets before any hope of developing them into drugs. Based on our findings, such drugs could have the potential to act alone or in combination with current immune-checkpoint therapies, reaching a large population of cancer patients.

In summary, we have discovered that exosomal PD-L1 is a major regulator of tumor progression through its ability to suppress T cell activation in draining lymph nodes and that its inhibition can lead to a long-lasting, systemic anti-tumor immunity.