Key Points This study has identified a novel capture mechanism for host-derived vesicles within the spleen and lymph node. This pathway modulates the immune response to circulating particulate antigens.

Exosomes are lipid-bound nanovesicles formed by inward budding of the endosomal membrane and released following fusion of the endosomal limiting membrane with the plasma membrane. We show here that primary leukocytes do not release exosomes unless subjected to potent activation signals, such as cytokine or mitogen stimulation. In particular, high levels of exosomes were released when murine splenic B cells were stimulated via CD40 and the IL-4 receptor. This property was shared by B cells from different anatomic locations, as newly formed, marginal zone and follicular B cells were capable of secreting exosomes upon CD40/IL-4 triggering. B cell exosomes expressed high levels of MHC class I, MHC class II, and CD45RA (B220), as well as components of the BCR complex, namely, surface Ig, CD19, and the tetraspanins CD9 and CD81. Ig on the plasma membrane of primary B cells was targeted to the exosome pathway, demonstrating a link between the BCR and this exocytic pathway. IgD and IgM were the predominant Ig isotypes associated with CD40/IL-4 elicited exosomes, though other isotypes (IgA, IgG1, IgG2a/2b, and IgG3) were also detected. Together, these results suggest that exosome release is not R constitutive activity of B cells, but may be induced following cell: cell signaling.

Extracellular vesicles (EV) are lipid particles released from eukaryotic cells into the extracellular fluid. Depending on the cell type or mechanism of release, vesicles vary in form and function and exert distinct functions in coagulation and immunity. Tumor cells may constitutively shed vesicles known as exosomes or microvesicles (MV). Alternatively, apoptosis induces the release of apoptotic blebs or vesicles (ApoV) from the plasma membrane. EV have been implicated in thrombotic events (the second highest cause of death in cancer patients) and tumor vesicles contribute to the anti-cancer immune response. In this study, we utilized the well characterized B16 melanoma model to determine the molecular composition and procoagulant and immunogenic potential of exosomes, MV and ApoV. Distinct patterns of surface and cytoplasmic molecules (tetraspanins, integrins, heat shock proteins and histones) were expressed between the vesicle types. Moreover, in vitro coagulation assays revealed that membrane-derived vesicles, namely MV and ApoV, were more procoagulant than exosomes–with tissue factor and phosphatidylserine critical for procoagulant activity. Mice immunized with antigen-pulsed ApoV and challenged with B16 tumors were protected out to 60 days, while lower protection rates were afforded by MV and exosomes. Together the results demonstrate distinct phenotypic and functional differences between vesicle types, with important procoagulant and immunogenic functions emerging for membrane-derived MV and ApoV versus endosome-derived exosomes. This study highlights the potential of EV to contribute to the prothrombotic state, as well as to anti-cancer immunity.

Apoptosis leads to the fragmentation and packaging of cellular contents into discrete vesicles, a process known as 'blebbing'. Extracellular vesicles express membrane-bound sialic acids, which enable their capture by CD169 (sialoadhesin; Siglec-1) expressing macrophages in the lymph node and spleen. Furthermore, CD169 mediates vesicle trafficking and suppresses the immune response to exosomes-a type of extracellular vesicle released from living cells. In this study, we found that CD169(+) macrophages were the predominant splenic macrophage subset responsible for the capture of EL4 lymphoma-derived apoptotic vesicles (ApoVs) from circulation. CD169(-/-) mice had significantly enhanced in vivo cytotoxic T lymphocyte responses to antigen-pulsed ApoVs, indicating a suppressive role for CD169(+) macrophages to ApoV-associated antigen. In contrast to the observed immunogenic role of ApoVs, the co-administration of unpulsed ApoVs with antigen-pulsed dendritic cells (DCs) significantly suppressed DC-mediated cytotoxic response in vivo; however, this occurred independent of CD169 expression. Overall, our results confirm that apoptosis contributes to both tolerance and immunity, as well as establishing CD169 as a critical mediator of the immune response to extracellular vesicles.

Increasing evidence suggests that NK cells act to promote effective T cell–based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria–dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.

Exosomes are lipid nanovesicles released after fusion of the endosomal limiting membrane with the plasma membrane. In this study, we investigated the requirement for CD4 T cells, B cells, and NK cells to provide help for CD8 T cell-mediated response to B cell-derived exosomes. CTL responses to Ag-loaded exosomes were dependent on host MHC class I, with a critical role for splenic langerin CD8α dendritic cells (DCs) in exosomal Ag cross-presentation. In addition, there was an absolute dependence on the presence of CD4 T cells, CD8 T cells, and NK cells, where the loss of any one of these subsets led to a complete loss of CTL response. Interestingly, NK cell depletion experiments demonstrated a critical cutoff point for depletion efficacy, with low-level residual NK cells providing sufficient help to allow optimal CD8 T cell proliferative responses to exosomal protein. Despite the potential role for B cells in the response to B cell-derived exosomal proteins, B cell depletion did not alter the exosome-induced CTL response. Similarly, a possible role for the BCR or circulating Ab in mediating CTL responses to B cell-derived exosomes was ruled out using DLMP2A mice, which lack secreted and membrane-bound Ab, yet harbor marginal zone and follicular B cells. In contrast, CTL responses to DC-derived exosomes were significantly inhibited within Ab-deficient DLMP2A mice compared with wild-type mice. However, this response was not restored upon serum transfer, implicating a role for the BCR, but not circulating Ab, in DC-derived exosome responses.

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Interferon-gamma (IFN-γ) is a critical cytokine for the initiation of immune responses against a variety of infectious agents and malignancies. We found that a range of Gram-positive and Gram-negative bacteria stimulated the rapid release (<24 h) of IFN-γ from murine leukocytes. Using fluorescence activated cell sorting and cd1d(-/-) and rag1(-/-) mice, we determined that dendritic cells (DCs) and natural killer (NK) cells were primarily responsible for IFN-γ release by Streptococcus salivarius, a Gram-positive commensal, previously noted to possess potent interleukin-12 (IL-12)-inducing potential. IFN-γ release from NK cells required DC:NK membrane contact and IL-12/IL-18 expression, but was independent of lymphocyte function-associated antigen-1-mediated interactions. IFN-γ release in response to bacteria was maintained in mice deficient for Toll-like receptor (TLR)-2 and TLR-4, suggesting that bacteria activate antigen-presenting cells via multiple, redundant pathways. Together, our results suggest that Gram-positive bacteria may be useful in driving NK cell activation and T helper 1 polarization and have the potential for development as effective adjuvants.