Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors

Systematically exploring the immune and housekeeping roles of the immunoproteasome

© 2012 Dr. Damien John ZankerA central role in infection-free immunity is the continual monitoring and surveillance for pathogens in the body. Every nucleated cell possesses the ability to display protein fragments (peptides) derived from the internal proteome coupled to major histocompatability (MHC) Class I complexes on their cell surface. This is a chief mechanism in place to alert immune cells such as CD8+ T cells (TCD8+) of their internal content and is integral in the detection of viruses and cancerous cells. However, only specialized cell subsets known as professional antigen presenting cells (pAPCs) are able to initiate a productive TCD8+ response. These cell types sample the environment they reside in and actively degrade these proteins for presentation to the immune system. Intracellular proteins are generally degraded by the proteasome; a cylindrical protein complex that contains catalytic β-subunits. In pAPC, an alternate subtype known as the ‘immunoproteasome’ exists. This is formed through substitution to an alternate set of β-subunits named LMP2, LMP7 and MECL-1. This specialised form has an altered cleavage motif and due to this, peptides created by immunoproteasomes are generally optimal for binding in a MHC Class I cleft. To date, a limited number of studies have demonstrated that the immunoproteasome may impact on TCD8+ repertoire and specific peptide processing. However, no studies have systematically assessed the contribution of individual immunoproteasome subunits to a whole immune response using naturally processed and presented peptides. Here, using genetically modified mouse strains that are deficient in one or two immunoproteasome subunits, we show that individual immunoproteasome subunits affect the TCD8+ repertoire to both dominant and subdominant responses in an influenza A virus (IAV) model. We further characterize the immunological functions of the immunoproteasome subunits through biochemical study of naturally processed and presented IAV peptides. We assess the rate at which epitope creation occurs in DCs derived from each of the immunoproteasome subunit-deficient mouse strains utilizing monospecificity TCD8+ specific for IAV epitopes and find further changes compared to wild type mice. As we find various IAV epitopes are presented at the cell surface at different times after infection, we are able to demonstrate that this presentation rate correlates with the immunodominance hierarchy displayed during secondary immune responses. During our studies we identify an interesting phenotype of lymphopenia in LMP2-/- mice. While the proteasome has been reported for other cellular functions including protein recycling, the central dogma for the immunoproteasome is that its sole function is proposed for antigen processing and presentation. Recent reports have outlined interesting immunoproteasome phenotypes associated with both mouse and human disorders, although mechanisms linking immunoproteasome function and disease have been limited. Here, we show that LMP2-deficiency impacts at key stages during T and B cell development. Using a variety of techniques, we identify a role for immunoproteasomal degradation in efficient NFκB signaling, which impacts on lymphocyte survival in LMP2-/- mice. Together, this thesis explores immunoproteasome function in regard to both the historical antigen processing and novel housekeeping roles of the immunoproteasome and clearly demonstrate its importance in overall cellular function

Resident CD8(+) and migratory CD103(+) dendritic cells control CD8 T cell immunity during acute influenza infection.

The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8(+) T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8(+) DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes

Memory regulatory T cells home to the lung and control influenza A virus infection

Memory regulatory T cells (mTregs) have been demonstrated to persist long-term in hosts after the resolution of primary influenza A virus (IAV) infection. However, whether such IAV infection-experienced (IAV-experienced) mTregs differentiate into a phenotypically and functionally distinct Treg subset and what function they play at the infection site remains poorly defined. In this study, we characterized the phenotype, examined the responsiveness and assessed the suppressive function of IAV-experienced memory Tregs (mTregs). In comparison with inexperienced naïve Tregs (nTregs), mTregs exhibited elevated expression of CD39, CD69, CD103, cytotoxic T lymphocyte-associated antigen-4, leukocyte function-associated antigen-1 and programmed cell death-1 and could be activated in an antigen-specific manner in vitro and in vivo. When mTregs and nTregs were adoptively cotransferred into recipient mice, mTregs had a competitive advantage in migrating to the IAV-infected lungs. mTregs were more capable of controlling in vitro proliferation of CD4+ and CD8+ T cells and suppressed CD40 and CD86 upregulation on bone marrow-derived dendritic cells. Adoptively transferred mTregs, but not adoptively transferred nTregs, significantly attenuated body weight loss, lung pathology and immune cell infiltration into the infected lungs after IAV infection. These results suggest that mTregs generated after IAV infection differentiate into a phenotypically distinct and functionally enhanced Treg subset that can be activated in an antigen-specific manner to exert immunosuppression. We propose vaccination to induce such mTregs as a potential novel strategy to protect against severe IAV infection

A Cancer Vaccine Induces Expansion of NY-ESO-1-Specific Regulatory T Cells in Patients with Advanced Melanoma

<div><p>Cancer vaccines are designed to expand tumor antigen-specific T cells with effector function. However, they may also inadvertently expand regulatory T cells (Treg), which could seriously hamper clinical efficacy. To address this possibility, we developed a novel assay to detect antigen-specific Treg based on down-regulation of surface CD3 following TCR engagement, and used this approach to screen for Treg specific to the NY-ESO-1 tumor antigen in melanoma patients treated with the NY-ESO-1/ISCOMATRIX^TM cancer vaccine. All patients tested had Treg (CD25^bright FoxP3⁺ CD127^neg) specific for at least one NY-ESO-1 epitope in the blood. Strikingly, comparison with pre-treatment samples revealed that many of these responses were induced or boosted by vaccination. The most frequently detected response was toward the HLA-DP4-restricted NY-ESO-1_157–170 epitope, which is also recognized by effector T cells. Notably, functional Treg specific for an HLA-DR-restricted epitope within the NY-ESO-1_115–132 peptide were also identified at high frequency in tumor tissue, suggesting that NY-ESO-1-specific Treg may suppress local anti-tumor immune responses. Together, our data provide compelling evidence for the ability of a cancer vaccine to expand tumor antigen-specific Treg in the setting of advanced cancer, a finding which should be given serious consideration in the design of future cancer vaccine clinical trials.</p> </div

Summary of NY-ESO-1-specific Treg responses detected in a cohort of 9 patients vaccinated with NY-ESO-1/ISCOMATRIX^TM vaccine.

<p>(<b><i>A</i></b>): For every patient within the cohort, each validated Treg response is summarized with a box. Responses are considered validated if they were observed in at least two independent cultures, using two independently synthesized batches of peptide. The position of the box indicates where in the NY-ESO-1 peptide sequence the response was localized. In the event that responses were detected to two peptides adjacent in sequence, this is shown as a single response spanning the two peptides. Shaded boxes indicate that the magnitude of the response was increased at least 2-fold in post-vaccination samples compared to pre-vaccination samples when both samples were tested in parallel under identical conditions. Solid boxes indicate that the response was only detectable in samples collected after vaccination. Open boxes indicate that the magnitude of the response was similar pre- and post-vaccination. (<b><i>B</i></b>): An example of a response that was induced by vaccination (Patient 124) is shown. Treg were gated on the basis of CD25 and FoxP3 expression, and CD3 down-regulation was assessed following re-stimulation with either control peptide or the same peptide used for expansion (NY-ESO-1_85–102).</p

Treg and Teff respond to an identical HLA-DP4-restricted epitope in the region NY-ESO-1_157–170.

<p>Patient PBMC were cultured for 21d with the 18mer peptide NY-ESO-1_157–174 and then re-stimulated with the indicated short HPLC-purified peptides by either adding directly to the culture as usual (<b><i>A–D</i></b>) or by pulsing onto BCL followed by washing (<b><i>E–F</i></b>). After overnight incubation, cells were stained and analyzed by flow cytometry, gating on Treg (CD4⁺ CD25⁺ FoxP3⁺; <b><i>A, C and E</i></b>) or Teff (CD4⁺ FoxP3⁻; <b><i>B, D and F</i></b>). Peptides used for re-stimulation were based on the published epitope NY-ESO-1_157–170, with either truncation (<b><i>A–B</i></b>) or extension (<b><i>C–D</i></b>) at each terminus. Graphs in <b><i>A–D</i></b> show results obtained for Patient 102; similar results were also obtained for Patient 103. Graphs in <b><i>E–F</i></b> show mean + SEM from Patients 102 and 113; an asterisk indicates a p value of <0.05 (t test).</p

Tumor tissue contains populations of NY-ESO-1-specific Treg and Teff.

<p>Tumour tissue was obtained from Patient 126 and TIL lines generated as described in <i>Methods</i>. (<b><i>A–B</i></b>): Cells were treated overnight with peptide NY-ESO-1_115–132 or control peptide, and then stained and analyzed by flow cytometry, gating on Treg (CD4⁺ CD25⁺ FoxP3⁺) or Teff (CD4⁺ FoxP3⁻) as indicated. Representative flow cytometry dot plots (<b>A</b>) show the gating of Treg and Teff, and the CD3 down-regulation response observed in each population following re-stimulation, while (<b>B</b>) shows a summary of results obtained in five experiments, each using one of the three different TIL lines generated. (<b><i>C</i></b>): The effect of blocking antibodies to HLA-DR, HLA-DP or HLA-DQ on the response to peptide NY-ESO-1_115–132 within Treg (left) and Teff (right) populations. Similar results were obtained in a second experiment. (<b><i>C</i></b>): TIL were stimulated overnight with NY-ESO-1_115–132 peptide and then peptide specific (CD3^lo) and non-specific (CD3^hi) Treg (CD4⁺ CD127^lo CD25^hi) were purified by cell sorting and tested for their ability to suppress the proliferation of CFSE-labeled CD8⁺ T cells pre-stimulated for 4hr with anti-CD3 at the indicated Treg:responder ratios. As a comparison, Treg were also sorted from previously frozen PBMC obtained from a healthy donor. Similar results were observed in a second experiment, although higher Treg:responder ratios were required to see suppression.</p