Melanoma growth and lymph node metastasis is independent of host CD169 expression.

Metastasis to the lymph node is a frequent and early event in tumour dissemination. Tumour soluble factors, including extracellular vesicles, condition host organs for metastatic tumour spread, thereby facilitating tumour cell migration and survival. In the peripheral lymphatics, extracellular vesicles are captured via their sialic acids by lymph node macrophages expressing the CD169 (sialoadhesin) molecule, thereby suppressing the immune response. We hypothesised that the CD169 molecule could modulate primary tumour growth and invasion into the regional lymph node by altering the immune response to tumour extracellular vesicles, or by directly interacting with invading tumour cells. No significant difference was noted in primary tumour growth between wild-type and CD169-/- mice, and protection against tumour challenge with tumour extracellular vesicle immunisation was similar between the strains. Subcutaneous implantation of B16 (F1 or F10) into the ventral-carpal aspect of forelimb resulted in melanoma infiltration into the axillary and brachial lymph nodes. CD169-/- mice displayed a lower level of metastatic lymph node lesions, however this failed to reach statistical significance. Although CD169 participates in the immune response to tumour antigen and appears to be a positive prognostic marker for human cancers, its role in modulating melanoma growth and metastasis is less clear.

Mechanistic insight into the procoagulant activity of tumor-derived apoptotic vesicles.

BACKGROUND: Chemotherapy induces the release of apoptotic vesicles (ApoV) from the tumor plasma membrane. Tumor ApoV may enhance the risk of thrombotic events in cancer patients undergoing chemotherapy. However, the relative contribution of ApoV to coagulation and the pathways involved remain poorly characterized. In addition, this study sets out to compare the procoagulant activity of chemotherapy-induced ApoV with their cell of origin and to determine the mechanisms of ApoV-induced coagulation. METHODS: We utilized human and murine cancer cell lines and chemotherapeutic agents to determine the requirement for the coagulation factors (tissue factor; TF, FII, FV, FVII, FVIII, FIX and phosphatidylserine) in the procoagulant activity of ApoV. The role of previously identified ApoV-associated FV was determined in a FV functional assay. RESULTS: ApoV were significantly more procoagulant per microgram of protein compared to parental living or dying tumor cells. In the phase to peak fibrin generation, procoagulant activity was dependent on phosphatidylserine, TF expression, FVII and the prothrombinase complex. However, the intrinsic coagulation factors FIX and FVIII were dispensable. ApoV-associated FV could not support coagulation in the absence of supplied, exogenous FV. CONCLUSIONS: ApoV are significantly more procoagulant than their parental tumor cells. ApoV require the extrinsic tenase and prothrombinase complex to activate the early phase of coagulation. Endogenous FV identified on tumor ApoV is serum-derived and functional, but is non-essential for ApoV-mediated fibrin generation. GENERAL SIGNIFICANCE: This study clarifies the mechanisms of procoagulant activity of vesicles released from dying tumor cells.

Procoagulant and immunogenic properties of melanoma exosomes, microvesicles and apoptotic vesicles.

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.

The CD169 sialoadhesin molecule mediates cytotoxic T-cell responses to tumour apoptotic vesicles.

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.

Altered transcription of murine genes induced in the small bowel by administration of probiotic strain Lactobacillus rhamnosus HN001.

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli (Lactobacillus-free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus-free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1, angptl4, and hspa1b, associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus-free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

NK cells are required for dendritic cell-based immunotherapy at the time of tumor challenge.

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.

CD169 mediates the capture of exosomes in spleen and lymph node.

Exosomes are lipid nanovesicles released following fusion of the endosoma limiting membrane with the plasma membrane; however, their fate in lymphoid organs after their release remains controversial. We determined that sialoadhesin (CD169; Siglec-1) is required for the capture of B cell-derived exosomes via their surface-expressed α2,3-linked sialic acids. Exosome-capturing macrophages were present in the marginal zone of the spleen and in the subcapsular sinus of the lymph node. In vitro assays performed on spleen and lymph node sections confirmed that exosome binding to CD169 was not solely due to preferential fluid flow to these areas. Although the circulation half-life of exosomes in blood of wild-type and CD169(-/-) mice was similar, exosomes displayed altered distribution in CD169(-/-) mice, with exosomes freely accessing the outer marginal zone rim of SIGN-R1(+) macrophages and F4/80(+) red pulp macrophages. In the lymph node, exosomes were not retained in the subcapsular sinus of CD169(-/-) mice but penetrated deeper into the paracortex. Interestingly, CD169(-/-) mice demonstrated an enhanced response to antigen-pulsed exosomes. This is the first report of a role for CD169 in the capture of exosomes and its potential to mediate the immune response to exosomal antigen.

Rapid interferon-gamma release from natural killer cells induced by a streptococcal commensal.

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.

Extracellular forms of Mycobacterium bovis BCG in the mucosal lymphatic tissues following oral vaccination.

Oral vaccination with BCG provides protective systemic immunity against pathogenic mycobacterial challenge. In this study, the anatomical distribution of Mycobacterium bovis BCG following oral vaccination was investigated. Replicating bacteria in the Peyer's patches and mesenteric lymph nodes were present as solitary rods or clusters of two to three bacteria, the majority of which were isolated ex vivo as extracellular forms. Only a minority were shown to be associated with typical antigen-presenting cells. Acid-fast staining of mast cell granules in lymphoid tissues revealed a potential pitfall for these analyses and may explain previous reports of acid-fast 'coccoid' forms of mycobacteria in tissues.

Streptokinase antibodies in patients presenting with acute coronary syndrome in three rural New Zealand populations.

BACKGROUND: New Zealand Maori have some of the highest rates of Group A streptococcal infection (GAS) in the world. GAS elevates titres of antistreptokinase (SK) neutralising antibodies and may induce resistance to SK. METHODS: Anti-SK titres were measured in 180 patients presenting with symptoms consistent with an acute coronary syndrome to three New Zealand rural hospitals, selected because they provide care for patients from communities with different socio-economic and ethnic mixes (Maori proportions varying between 6% and 67%). FINDINGS: Compared with the community with the lowest proportion of Maori, patients in the community with the highest proportion of Maori had mean anti-SK titres that were 2.8 times higher (p=0.05). They were 2.5 times more likely to have a high anti-SK titre (33% vs 13% p=0.035). INTERPRETATION: Alternatives to reperfusion with SK should be the first-choice therapy in hospitals serving communities with high rates of GAS such as some predominantly Maori and Pacific Island communities.

Urinary soluble HLA-DR is a potential biomarker for acute renal transplant rejection.

BACKGROUND.: Urine is a potentially rich source of biomarkers for monitoring kidney dysfunction. In this study, we have investigated the potential of soluble human leukocyte antigen (sHLA)-DR in the urine for noninvasive monitoring of renal transplant patients. METHODS.: Urinary soluble HLA-DR levels were measured by sandwich enzyme-linked immunosorbent assay in 103 patients with renal diseases or after renal transplantation. sHLA-DR in urine was characterized by Western blotting and mass spectrometry. RESULTS.: Acute graft rejection was associated with a significantly elevated level of urinary sHLA-DR (P<0.0001), compared with recipients with stable graft function or healthy individuals. A receiver operating characteristic curve analysis showed the area under the curve to be 0.88 (P<0.001). At a selected threshold, the sensitivity was 80% and specificity was 98% for detection of acute renal transplant rejection. sHLA-DR was not exosomally associated and was of lower molecular weight compared with the HLA-DR expressed as heterodimer on the plasma membrane of antigen-presenting cells. CONCLUSIONS.: sHLA-DR excreted into urine is a promising indicator of renal transplant rejection.

Induction of exosome release in primary B cells stimulated via CD40 and the IL-4 receptor.

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 constitutive activity of B cells, but may be induced following cell: cell signaling.

Lymphatic tracing and T cell responses following oral vaccination with live Mycobacterium bovis (BCG).

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis bacille Calmette-Guérin (BCG) expands a subset of interferon-gamma (IFN-gamma)-secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyer's patches. Lymphocytes that produced IFN-gamma in response to PPD-B or BCG-pulsed dendritic cells predominated in the spleen and were almost exclusively CD4(+), CD44(+) and CD62L(-), thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN-gamma-secreting T cells in vaccinated mice did not express the mucosal homing antigens alpha(4)beta(7) integrin or alphaIEL (CD103). However, a proportion of BCG-specific CD4(+) T cells expressed the CD29 integrin (beta(1)) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen-resident CD4(+) T cells with the potential to provide protective immunity in the lung.

Circulating, soluble forms of major histocompatibility complex antigens are not exosome-associated.

In vitro studies have shown that soluble MHC (sMHC) released by cell lines is bound to nano-vesicles termed exosomes. It is thought that exosomes may represent the major reservoir of sMHC class I and II molecules in biological fluids. However, most studies have been confined to in vitro assays performed with cell lines. We show here that sMHC in the serum or plasma differs from exosome-bound sMHC in five ways: In contrast to exosome-associated sMHC, circulating sMHC is of low density, has a low apparent molecular mass (40-300 kDa) and is not detergent-labile. Moreover, the majority of MHC class II isoforms and MHC class I in blood are not physically linked and circulating HLA-DR is accessible to an antibody specific for the HLA-DR alpha-chain intracellular epitope, which is masked by its association with cellular or exosomal membranes. Finally, utilizing transcriptional activator of murine MHC class II (C2ta) promoter-mutant mice, we showed that the release of sMHC class II into the circulation is dependent on the C2ta pI promoter, but not pIII or pIV. This suggests that myeloid dendritic cells and/or macrophages, which preferentially use promoter pI of the C2ta gene, produce most of the sMHC class II found in the circulation.