Vagus Nerve Stimulation Modulates Inflammation in Treatment-Resistant Depression Patients: A Pilot Study.

Vagal neurostimulation (VNS) is used for the treatment of epilepsy and major medical-refractory depression. VNS has neuropsychiatric functions and systemic anti-inflammatory activity. The objective of this study is to measure the clinical efficacy and impact of VNS modulation in depressive patients. Six patients with refractory depression were enrolled. Depression symptoms were assessed with the Montgomery-Asberg Depression Rating, and anxiety symptoms with the Hamilton Anxiety Rating Scale. Plasmas were harvested prospectively before the implantation of VNS (baseline) and up to 4 years or more after continuous therapy. Forty soluble molecules were measured in the plasma by multiplex assays. Following VNS, the reduction in the mean depression severity score was 59.9% and the response rate was 87%. Anxiety levels were also greatly reduced. IL-7, CXCL8, CCL2, CCL13, CCL17, CCL22, Flt-1 and VEGFc levels were significantly lowered, whereas bFGF levels were increased (p values ranging from 0.004 to 0.02). This exploratory study is the first to focus on the long-term efficacy of VNS and its consequences on inflammatory biomarkers. VNS may modulate inflammation via an increase in blood-brain barrier integrity and a reduction in inflammatory cell recruitment. This opens the door to new pathways involved in the treatment of refractory depression.

Fecal microbiota transplantation plus anti-PD-1 immunotherapy in advanced melanoma: a phase I trial.

Fecal microbiota transplantation (FMT) represents a potential strategy to overcome resistance to immune checkpoint inhibitors in patients with refractory melanoma; however, the role of FMT in first-line treatment settings has not been evaluated. We conducted a multicenter phase I trial combining healthy donor FMT with the PD-1 inhibitors nivolumab or pembrolizumab in 20 previously untreated patients with advanced melanoma. The primary end point was safety. No grade 3 adverse events were reported from FMT alone. Five patients (25%) experienced grade 3 immune-related adverse events from combination therapy. Key secondary end points were objective response rate, changes in gut microbiome composition and systemic immune and metabolomics analyses. The objective response rate was 65% (13 of 20), including four (20%) complete responses. Longitudinal microbiome profiling revealed that all patients engrafted strains from their respective donors; however, the acquired similarity between donor and patient microbiomes only increased over time in responders. Responders experienced an enrichment of immunogenic and a loss of deleterious bacteria following FMT. Avatar mouse models confirmed the role of healthy donor feces in increasing anti-PD-1 efficacy. Our results show that FMT from healthy donors is safe in the first-line setting and warrants further investigation in combination with immune checkpoint inhibitors. ClinicalTrials.gov identifier NCT03772899 .

T cell-loaded injectable chitosan scaffold shows short-term efficacy in localised cancer immunotherapy in mice.

Adoptive cell therapy (ACT) shows success against treatment-resistant cancers, but is limited by the large number of intravenously delivered T cells required and toxicity related to systemic administration. In this work, we hypothesized that localized T cell delivery in an in situ gelling chitosan hydrogel will allow similar treatment efficacy despite delivering fewer cells than systemic intravenous delivery. A rapidly gelling chitosan gel with good mechanical properties was used for this study. Gel biocompatibility and biodegradability were tested over 8 weeks in mice. No adverse effects were observed. The gel elicited a local granulomatous reaction (foreign body reaction), degrading by about 75% volume at 8 weeks. The survival, escape and bioactivity against the tumour cells of encapsulated murine lymphocytes (OT-I) and human Jurkat cells were confirmed in vitro by live/dead assay and flow cytometry. Efficacy was studied using a mouse tumour model where the injected OT-I can specifically recognize and attack ovalbumin (OVA) protein-expressing tumours. The OT-I cell delivery scaffold was compared to untreated controls, OT-I in saline and intravenous systemic treatment with 3-fold more OT-I, observing tumour growth and localization by intravital microscopy and histology. Gel-encapsulated OT-I limited tumour growth significantly up to 11 days after treatment compared to that of untreated mice and mice with longer PBS-suspended OT-I treatment (9 days), but slightly less than that of mice with IV-delivered OT-I treatment (14 days). No significant difference was observed when directly comparing the gel and IV treatments. Although further optimization of the treatment is required, this work shows the feasibility and potential of the chitosan gel for localised OT-I delivery in cancer immunotherapy.

Blood Sample Processing and Banking for Functional and Molecular Analyses.

Immune monitoring of circulating immune cells in the blood provides insight into a patient's own immune response over the course of a treatment or disease progression. Information such as whether immune cells are functional or non-functional and what specific proteins they express or secrete can be essential to understand if (and how) a treatment is working or a disease is progressing. To do so, it requires careful handling and storage of precious biological samples with the goals of obtaining a large amount of information from limited samples and minimizing future research costs by the use of banked samples. Many factors, including blood sample types, time of collection, containers used, preservatives and other additives, transport means, and length of transit time, all affect the quality of the samples and the stability of biomarkers and must be considered at the initial collection stage. An efficient study design includes provisions for further processing of the original samples, such as cryopreservation of isolated cells, purification of DNA and RNA, and preparation of specimens for genomic, immunological, and biochemical analyses. Development of standard operating procedures and quality control plans is a safeguard of the samples' quality and of the validity of the results. Here, we focus on the collection and processing of blood suitable for plasma and peripheral blood mononuclear cell (PBMC) banking, including collection, processing, and storage of samples, based on our experience.

Biomaterials for enhanced immunotherapy.

Cancer immunotherapies have revolutionized the treatment of numerous cancers, with exciting results often superior to conventional treatments, such as surgery and chemotherapy. Despite this success, limitations such as limited treatment persistence and toxic side effects remain to be addressed to further improve treatment efficacy. Biomaterials offer numerous advantages in the concentration, localization and controlled release of drugs, cancer antigens, and immune cells in order to improve the efficacy of these immunotherapies. This review summarizes and highlights the most recent advances in the use of biomaterials for immunotherapies including drug delivery and cancer vaccines, with a particular focus on biomaterials for immune cell delivery.

Phosphorylation of eIF4E in the stroma drives the production and spatial organisation of collagen type I in the mammary gland.

The extracellular matrix (ECM) plays critical roles in breast cancer development. Whether ECM composition is regulated by the phosphorylation of eIF4E on serine 209, an event required for tumorigenesis, has not been explored. Herein, we used proteomics and mouse modeling to investigate the impact of mutating serine 209 to alanine on eIF4E (i.e., S209A) on mammary gland (MG) ECM. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD028953. We discovered that S209A knock-in mice, expressing a non-phosphorylatable form of eIF4E, have less collagen-I deposition in native and tumor-bearing MGs, leading to altered tumor cell invasion. Additionally, phospho-eIF4E deficiency impacts collagen topology; fibers at the tumor-stroma boundary in phospho-eIF4E-deficient mice run parallel to the tumor edge but radiate outwards in wild-type mice. Finally, a phospho-eIF4E-deficient tumor microenvironment resists anti-PD-1 therapy-induced collagen deposition, correlating with an increased anti-tumor response to immunotherapy. Clinically, we showed that collagen-I and phospho-eIF4E are positively correlated in human breast cancer samples, and that stromal phospho-eIF4E expression is influenced by tumor proximity. Together, our work defines the importance of phosphorylation of eIF4E on S209 as a regulator of MG collagen architecture in the tumor microenvironment, thereby positioning phospho-eIF4E as a therapeutic target to augment response to therapy.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

Single-dose replicating poxvirus vector-based RBD vaccine drives robust humoral and T cell immune response against SARS-CoV-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

MHC class I antigen cross-presentation mediated by PapMV nanoparticles in human antigen-presenting cells is dependent on autophagy.

Nanoparticles made of the coat protein of papaya mosaic virus (PapMV) and a single-strand RNA were previously shown to be an efficient antigen presentation system for the trigger of cellular immunity. Engineering of PapMV nano with a cytotoxic T lymphocyte epitope was previously shown activating specific T lymphocytes through a proteasome-independent major histocompatibility complex class I (MHC-I) cross-presentation. In this study, we provide new insights into the mechanism of the MHC-I cross-presentation mediated by PapMV nanoparticles. We demonstrate that PapMV nanoparticles do not require the transporter associated with antigen presentation (TAP), but rather depend on lysosome acidification and cathepsin S protease activity for presentation of the T cell epitope. We have also linked the induction of autophagy with this vacuolar MHC-I cross-presentation process. Interestingly, autophagy is induced in antigen-presenting cells after PapMV nanoparticles exposure and inhibition of autophagy reduce MHC-I cross-presentation. This study demonstrates that autophagy is associated with TAP- and proteasome-independent MHC-I cross-presentation. A deeper understanding of the autophagy-dependent MHC-I cross-presentation will be useful in designing vaccination platforms that aim to trigger an efficient cytotoxic T lymphocyte response.

CD40L-Stimulated B Lymphocytes Are Polarized toward APC Functions after Exposure to IL-4 and IL-21.

B lymphocytes have multiple functions central to humoral immunity, including Ag presentation to T cells, cytokine secretion, and differentiation into Ab-secreting plasma cells. In vitro expansion of human B cells by continuous IL-4 stimulation and engagement of their CD40 receptor by CD40L has allowed the use of these IL-4-CD40-B cells in research for the induction of Ag-specific T cell immune responses. However, in vivo, follicular helper T cells also influence B cell activity through the secretion of IL-21. The impact of both cytokines on multiple B cell functions is not clearly defined. To further understand these cytokines in CD40-B cell biology, we stimulated CD40-B cells with IL-4 or IL-21 or both (Combo) and characterized the proliferation, subsets, and functions of these cells. We demonstrate that IL-21- and Combo-CD40-B cells are highly proliferative cells that can be rapidly expanded to high numbers. We show that IL-21-CD40-B cells polarize to Ab-secreting plasma cells, whereas IL-4- and Combo-CD40-B cells are mostly activated mature B cells that express molecules associated with favorable APC functions. We further demonstrate that both IL-4- and Combo-CD40-B cells are efficient in promoting T cell activation and proliferation compared with IL-21-CD40-B cells. Thus, our study provides a better appreciation of CD40-B cell plasticity and biology. In addition, the stimulation of B cells with CD40L, IL-4, and IL-21 allows for the fast generation of high numbers of efficient APC, therefore providing a prospective tool for research and clinical applications such as cancer immunotherapy.

The MNK1/2-eIF4E Axis Supports Immune Suppression and Metastasis in Postpartum Breast Cancer.

Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4ES209A) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8+ T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy.

High-dimensional analysis of the adenosine pathway in high-grade serous ovarian cancer.

BACKGROUND: Hydrolysis of extracellular ATP to adenosine (eADO) is an important immune checkpoint in cancer immunology. We here investigated the impact of the eADO pathway in high-grade serous ovarian cancer (HGSC) using multiparametric platforms. METHODS: We performed a transcriptomic meta-analysis of eADO-producing CD39 and CD73, an eADO signaling gene signature, immune gene signatures and clinical outcomes in approximately 1200 patients with HGSC. Protein expression, localization and prognostic impact of CD39, CD73 and CD8 were then performed on approximately 1000 cases on tissue microarray, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry and single-cell RNA sequencing on a subset of patients. RESULTS: Concomitant CD39 and CD73 gene expression, as well as high levels of an eADO gene signature, were associated with worse prognosis in patients with HGSC, notably in the immunoregulatory molecular subtype, characterized by an immune-active microenvironment. CD39 was further associated with primary chemorefractory and chemoresistant human HGSC and platinum-based chemotherapy of murine HGSC was significantly more effective in CD39-deficient mice. At protein level, CD39 and CD73 were predominantly expressed by cancer-associated fibroblasts, and CD39 was expressed on severely exhausted, clonally expanded and putative tissue-resident memory TILs. CONCLUSIONS: Our study revealed the clinical, immunological, subtype-specific impacts of eADO signaling in HGSC, unveiled the chemoprotective effect of CD39 and supports the evaluation of eADO-targeting agents in patients with ovarian cancer.

A Randomized Controlled Study to Evaluate the Safety and Reactogenicity of a Novel rVLP-Based Plant Virus Nanoparticle Adjuvant Combined with Seasonal Trivalent Influenza Vaccine Following Single Immunization in Healthy Adults 18-50 Years of Age.

Inactivated influenza vaccines efficacy is variable and often poor. We conducted a phase 1 trial (NCT02188810), to assess the safety and immunogenicity of a novel nanoparticle Toll-like receptor 7/8 agonist adjuvant (Papaya Mosaic Virus) at different dose levels combined with trivalent influenza vaccine in healthy persons 18-50 years of age. Hemagglutination-inhibition assays, antibody to Influenza A virus nucleoprotein and peripheral blood mononuclear cells for measurement of interferon-gamma ELISPOT response to influenza antigens, Granzyme B and IFNγ:IL-10 ratio were measured. The most common adverse events were transient mild to severe injection site pain and no safety signals were observed. A dose-related adjuvant effect was observed. Geometric mean hemagglutination-inhibition titers increased at day 28 in most groups and waned over time, but fold-antibody responses were poor in all groups. Cell mediated immunity results were consistent with humoral responses. The Papaya Mosaic Virus adjuvant in doses of 30 to 240 µg combined with reduced influenza antigen content was safe with no signals up to 3 years after vaccination. A dose-related adjuvant effect was observed and immunogenicity results suggest that efficacy study should be conducted in influenza antigen-naïve participants.

Founder BRCA1/BRCA2/PALB2 pathogenic variants in French-Canadian breast cancer cases and controls.

Inherited germline pathogenic variants are responsible for ~5% of breast cancer globally. Through rapid expansion and isolation since immigration in the early 17th century, French Canadians are a relatively genetically homogenous founder population and therefore represent a unique demographic for genetic contributions to disease. To date, twenty variants in BRCA1, BRCA2, and PALB2 that predispose families to breast and ovarian cancer have been identified as recurring in the French-Canadian founder population. Our objective was to evaluate the clinical efficacy and validity of targeted genetic testing for these variants in Montreal French Canadians. A total of 555 breast cancer cases unselected for family history or age of diagnosis were genotyped, along with 1940 controls without a personal or family history of cancer. A Sequenom genotyping assay identified a pathogenic variant in 0.2% (5 of 1940) of cancer-free controls, and 3.8% (21/555) of breast cancer cases. Almost 10% (12/113) of early onset cases were heterozygous for founder BRCA1 or BRCA2 pathogenic variant. Of twenty variants tested, only seven were identified in this study. The option of providing this test as population-based screening is discussed.

Moving towards personalized treatments of immune-related adverse events.

The enhancement of immune responses upon treatment with immune checkpoint inhibitors can have the desired outcome of reinvigorating antitumour immune surveillance, but often at the expense of immune-related adverse events (irAEs). This novel disease entity often prompts comparisons with, and extrapolation of treatment approaches from, primary autoimmune disorders. Accordingly, current treatment guidelines for irAEs make generic recommendations adapted from the literature describing primary autoimmune diseases, without taking into consideration the substantial disparity of the immunohistopathological findings within each organ affected by an irAE. The treatment modalities themselves are complex and have many potential drawbacks, such as serious and rarely fatal infections, drug toxicities overlapping with irAEs and the risk of compromising cancer immune surveillance. Herein, we provide an overview of key cellular and soluble immunological factors mediating irAEs and propose a model integrating this knowledge with the immunohistopathological findings of the affected organs for a personalized decision-making process for each patient.

Innate immunity activation in the early brain injury period following subarachnoid hemorrhage.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (SAH) is a catastrophic disease with devastating consequences, including a high mortality rate and severe disabilities among survivors. Inflammation is induced following SAH, but the exact role and phenotype of innate immune cells remain poorly characterized. We investigated the inflammatory components of the early brain injury in an animal model and in SAH patients. METHOD: SAH was induced through injection of blood in the subarachnoid space of C57Bl/6 J wild-type mice. Prospective blood collections were obtained at 12 h, days 1, 2, and 7 to evaluate the systemic inflammatory consequences of SAH by flow cytometry and enzyme-linked immunosorbent-assay (ELISA). Brains were collected, enzymatically digested, or fixed to characterize infiltrating inflammatory cells and neuronal death using flow cytometry and immunofluorescence. Phenotypic evaluation was performed at day 7 using the holding time and footprint tests. We then compared the identified inflammatory proteins to the profiles obtained from the plasma of 13 human SAH patients. RESULTS: Following SAH, systemic IL-6 levels increased rapidly, whereas IL-10 levels were reduced. Neutrophils were increased both in the brain and in the blood reflecting local and peripheral inflammation following SAH. More intracerebral pro-inflammatory monocytes were found at early time points. Astrocyte and microglia activation were also increased, and mice had severe motor deficits, which were associated with an increase in the percentage of caspase-3-positive apoptotic neurons. Similarly, we found that IL-6 levels in patients were rapidly increased following SAH. ICAM-1, bFGF, IL-7, IL-12p40, and MCP-4 variations over time were different between SAH patients with good versus bad outcomes. Moreover, high levels of Flt-1 and VEGF at admission were associated with worse outcomes. CONCLUSION: SAH induces an early intracerebral infiltration and peripheral activation of innate immune cells. Furthermore, microglia and astrocytic activation are present at later time points. Our human and mouse data illustrate that SAH is a systemic inflammatory disease and that immune cells represent potential therapeutic targets to help this population of patients in need of new treatments.

Targeting the mTOR pathway uncouples the efficacy and toxicity of PD-1 blockade in renal transplantation.

Immune checkpoint inhibitor (ICI) use remains a challenge in patients with solid organ allografts as most would undergo rejection. In a melanoma patient in whom programmed-death 1 (PD-1) blockade resulted in organ rejection and colitis, the addition of the mTOR inhibitor sirolimus resulted in ongoing anti-tumor efficacy while promoting allograft tolerance. Strong granzyme B+, interferon (IFN)-γ+ CD8+ cytotoxic T cell and circulating regulatory T (Treg) cell responses were noted during allograft rejection, along with significant eosinophilia and elevated serum IL-5 and eotaxin levels. Co-treatment with sirolimus abated cytotoxic T cell numbers and eosinophilia, while elevated Treg cell numbers in the peripheral blood were maintained. Interestingly, numbers of IFN-γ+ CD4+ T cells and serum IFN-γ levels increased with the addition of sirolimus treatment likely promoting ongoing anti-PD-1 efficacy. Thus, our results indicate that sirolimus has the potential to uncouple anti-PD-1 therapy toxicity and efficacy.

Immune-enrichment of non-small cell lung cancer baseline biopsies for multiplex profiling define prognostic immune checkpoint combinations for patient stratification.

BACKGROUND: Permanence of front-line management of lung cancer by immunotherapies requires predictive companion diagnostics identifying immune-checkpoints at baseline, challenged by the size and heterogeneity of biopsy specimens. METHODS: An innovative, tumor heterogeneity reducing, immune-enriched tissue microarray was constructed from baseline biopsies, and multiplex immunofluorescence was used to profile 25 immune-checkpoints and immune-antigens. RESULTS: Multiple immune-checkpoints were ranked, correlated with antigen presenting and cytotoxic effector lymphocyte activity, and were reduced with advancing disease. Immune-checkpoint combinations on TILs were associated with a marked survival advantage. Conserved combinations validated on more than 11,000 lung, breast, gastric and ovarian cancer patients demonstrate the feasibility of pan-cancer companion diagnostics. CONCLUSIONS: In this hypothesis-generating study, deepening our understanding of immune-checkpoint biology, comprehensive protein-protein interaction and pathway mapping revealed that redundant immune-checkpoint interactors associate with positive outcomes, providing new avenues for the deciphering of molecular mechanisms behind effects of immunotherapeutic agents targeting immune-checkpoints analyzed.