Nociceptor neurons affect cancer immunosurveillance.

Solid tumours are innervated by nerve fibres that arise from the autonomic and sensory peripheral nervous systems1-5. Whether the neo-innervation of tumours by pain-initiating sensory neurons affects cancer immunosurveillance remains unclear. Here we show that melanoma cells interact with nociceptor neurons, leading to increases in their neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. Calcitonin gene-related peptide (CGRP)-one such nociceptor-produced neuropeptide-directly increases the exhaustion of cytotoxic CD8+ T cells, which limits their capacity to eliminate melanoma. Genetic ablation of the TRPV1 lineage, local pharmacological silencing of nociceptors and antagonism of the CGRP receptor RAMP1 all reduced the exhaustion of tumour-infiltrating leukocytes and decreased the growth of tumours, nearly tripling the survival rate of mice that were inoculated with B16F10 melanoma cells. Conversely, CD8+ T cell exhaustion was rescued in sensory-neuron-depleted mice that were treated with local recombinant CGRP. As compared with wild-type CD8+ T cells, Ramp1-/- CD8+ T cells were protected against exhaustion when co-transplanted into tumour-bearing Rag1-deficient mice. Single-cell RNA sequencing of biopsies from patients with melanoma revealed that intratumoral RAMP1-expressing CD8+ T cells were more exhausted than their RAMP1-negative counterparts, whereas overexpression of RAMP1 correlated with a poorer clinical prognosis. Overall, our results suggest that reducing the release of CGRP from tumour-innervating nociceptors could be a strategy to improve anti-tumour immunity by eliminating the immunomodulatory effects of CGRP on cytotoxic CD8+ T cells.

Navigating the blurred path of mixed neuroimmune signaling.

Evolution has created complex mechanisms to sense environmental danger and protect tissues, with the nervous and immune systems playing pivotal roles. These systems work together, coordinating local and systemic reflexes to restore homeostasis in response to tissue injury and infection. By sharing receptors and ligands, they influence the pathogenesis of various diseases. Recently, a less-explored aspect of neuroimmune communication has emerged: the release of neuropeptides from immune cells and cytokines/chemokines from sensory neurons. This article reviews evidence of this unique neuroimmune interplay and its impact on the development of allergy, inflammation, itch, and pain. We highlight the effects of this neuroimmune signaling on vital processes such as host defense, tissue repair, and inflammation resolution, providing avenues for exploration of the underlying mechanisms and therapeutic potential of this signaling.

An engineered Accum-E7 protein-based vaccine with dual anti-cervical cancer activity.

Worldwide prevalence of cervical cancer decreased significantly with the use of human papilloma virus (HPV)-targeted prophylactic vaccines. However, these multivalent antiviral vaccines are inert against established tumors, which leave patients with surgical ablative options possibly resulting in long-term reproductive complications and morbidity. In an attempt to bypass this unmet medical need, we designed a new E7 protein-based vaccine formulation using Accum™, a technology platform designed to promote endosome-to-cytosol escape as a means to enhance protein accumulation in target cells. Prophylactic vaccination of immunocompetent mice using the Accum-E7 vaccine (aE7) leads to complete protection from cervical cancer despite multiple challenges conducted with ascending C3.43 cellular doses (0.5-, 1.0-, and 2.0 × 106 cells). Moreover, the humoral response induced by aE7 was higher in magnitude compared with naked E7 protein vaccination and displayed potent inhibitory effects on C3.43 proliferation in vitro. When administered therapeutically to animals with pre-established C3.43 or Tal3 tumors, the vaccine-induced response synergized with multiple immune checkpoint blockers (anti-PD-1, anti-CTLA4, and anti-CD47) to effectively control tumor growth. Mechanistically, the observed therapeutic effect requires cross-presenting dendritic cells as well as CD8 T cells predominantly, with a non-negligible role played by both CD4+ and CD19+ lymphocytes. good laboratory practice (GLP) studies revealed that aE7 is immunogenic and well tolerated by immunocompetent mice with no observed adverse effects despite the use of a fourfold exceeding dose. In a nutshell, aE7 represents an ideal vaccine candidate for further clinical development as it uses a single engineered protein capable of exhibiting both prophylactic and therapeutic activity.

Local delivery of accutox® synergises with immune-checkpoint inhibitors at disrupting tumor growth.

BACKGROUND: The Accum® platform was initially designed to accumulate biomedicines in target cells by inducing endosomal-to-cytosol escape. Interestingly however, the use of unconjugated Accum® was observed to trigger cell death in a variety of cancer cell lines; a property further exploited in the development of Accum®-based anti-cancer therapies. Despite the impressive pro-killing abilities of the parent molecule, some cancer cell lines exhibited resistance. This prompted us to test additional Accum® variants, which led to the identification of the AccuTOX® molecule. METHODS: A series of flow-cytometry and cell-based assays were used to assess the pro-killing properties of AccuTOX® along with its ability to trigger the production of reactive oxygen species (ROS), endosomal breaks and antigen presentation. RNA-seq was also conducted to pinpoint the most prominent processes modulated by AccuTOX® treatment in EL4 T-cell lymphoma. Finally, the therapeutic potency of intratumorally-injected AccuTOX® was evaluated in three different murine solid tumor models (EL4, E0771 and B16) both as a monotherapy or in combination with three immune-checkpoint inhibitors (ICI). RESULTS: In total, 7 Accum® variants were screened for their ability to induce complete cell death in 3 murine (EL4, B16 and E0771) and 3 human (MBA-MD-468, A549, and H460) cancer cell lines of different origins. The selected compound (hereafter refereed to as AccuTOX®) displayed an improved killing efficiency (~ 5.5 fold compared to the parental Accum®), while retaining its ability to trigger immunogenic cell death, ROS production, and endosomal breaks. Moreover, transcriptomic analysis revealed that low dose AccuTOX® enhances H2-Kb cell surface expression as well as antigen presentation in cancer cells. The net outcome culminates in impaired T-cell lymphoma, breast cancer and melanoma growth in vivo especially when combined with anti-CD47, anti-CTLA-4 or anti-PD-1 depending on the animal model. CONCLUSIONS: AccuTOX® exhibits enhanced cancer killing properties, retains all the innate characteristics displayed by the parental Accum® molecule, and synergizes with various ICI in controlling tumor growth. These observations will certainly pave the path to continue the clinical development of this lead compound against multiple solid tumor indications.

Intratumoral administration of unconjugated Accum™ impairs the growth of pre-established solid lymphoma tumors.

The Accum™ technology was initially designed to enhance the bioaccumulation of a given molecule in target cells. It does so by triggering endosomal membrane damages allowing endocytosed products to enter the cytosol, escaping the harsh environmental cues of the endosomal lumen. In an attempt to minimize manufacturing hurdles associated with Accum™ conjugation, we tested whether free Accum™ admixed with antigens could lead to outcomes similar to those obtained with conjugated products. Surprisingly, unconjugated Accum™ was found to promote cell death in vitro, an observation further confirmed on various murine tumor cell lines (EL4, CT-26, B16, and 4 T1). At the molecular level, unconjugated Accum™ triggers the production of reactive oxygen species and elicits immunogenic cell death while retaining its innate ability to cause endosomal damages. When administered as a monotherapy to animals with pre-established EL4 T-cell lymphoma, Accum™ controlled tumor growth in a dose-dependent manner, and its therapeutic effect relies on CD4 and CD8 T cells. Although unconjugated Accum™ synergizes with various immune checkpoint inhibitors (anti-CTLA4, anti-PD-1, or anti-CD47) at controlling tumor growth, its therapeutic potency could not be further enhanced when combined with all three tested immune checkpoint inhibitors at once due to its dependency on a specific dosing regimen. In sum, we report in this study an unprecedented new function for unconjugated Accum™ as a novel anticancer molecule. These results could pave the path for a new line of investigation aimed at exploring the pro-killing properties of additional Accum™ variants as a mean to develop second-generation anticancer therapeutics.

Bone marrow aspirate concentrate versus platelet-rich plasma for treating knee osteoarthritis: a one-year non-randomized retrospective comparative study.

BACKGROUND: Knee osteoarthritis (OA) is a debilitating condition affecting human body biomechanics and quality of life. Current standard care for knee OA leads to trivial improvement and entails multiple adverse effects or complications. Recently, investigational cell therapies injected intra-articularly, such as bone marrow aspirate concentrate (BMAC) and platelet-rich plasma (PRP), have shown safety and therapeutic potency providing patients with pain relief. In the current retrospective comparative study, we investigated the differences in pain and functional improvements in patients with symptomatic knee OA receiving intra-articular injections of BMAC vs PRP. METHODS: Pain and functionality scores were measured at baseline and at different time points post-injection over 12 months, using 3 self-administered, clinically validated questionnaires: the visual analogue scale (VAS) for assessing pain intensity, the knee injury and osteoarthritis outcome score (KOOS) for evaluating functionality and knee-related quality of life, and the Western Ontario and McMaster Universities Arthritis Index (WOMAC) for evaluating physical function. The repeated-measures general linear model with Sidak test for pairwise comparisons was used to investigate the influence of the treatment on the score evolution within groups (between baseline and each time point) and between groups (overall). RESULTS: The BMAC group (n = 26 knees) significantly improved in VAS, KOOS, and WOMAC scores between baseline and 12 months (57.4, 75.88, and 73.95% mean score improvement, respectively). In contrast, the PRP group (n = 13 knees) witnessed nonsignificant improvement in all scores. BMAC, in comparison to PRP, induced significant improvement in outcomes by 29.38% on the VAS scale, 53.89% on the KOOS scale, and 51.71% on the WOMAC scale (P < .002, P < .01, P < .011, respectively). CONCLUSIONS: Intra-articular autologous BMAC injections are safe, effective in treating pain, and ameliorate functionality in patients with symptomatic knee OA to a greater extent than PRP injections. Intra-articular autologous BMAC therapy is safe and provides more relief to patients with symptomatic knee osteoarthritis compared to PRP therapy.

FcεR1-expressing nociceptors trigger allergic airway inflammation.

BACKGROUND: Lung nociceptor neurons amplify immune cell activity and mucus metaplasia in response to an inhaled allergen challenge in sensitized mice. OBJECTIVE: We sought to identify the cellular mechanisms by which these sensory neurons are activated subsequent to allergen exposure. METHODS: We used calcium microscopy and electrophysiologic recording to assess whether vagal neurons directly respond to the model allergen ovalbumin (OVA). Next, we generated the first nociceptor-specific FcεR1γ knockdown (TRPV1Cre::FcεR1γfl/fl) mice to assess whether this targeted invalidation would affect the severity of allergic inflammation in response to allergen challenges. RESULTS: Lung-innervating jugular nodose complex ganglion neurons express the high-affinity IgE receptor FcεR1, the levels of which increase in OVA-sensitized mice. FcεR1γ-expressing vagal nociceptor neurons respond directly to OVA complexed with IgE with depolarization, action potential firing, calcium influx, and neuropeptide release. Activation of vagal neurons by IgE-allergen immune complexes, through the release of substance P from their peripheral terminals, directly amplifies TH2 cell influx and polarization in the airways. Allergic airway inflammation is decreased in TRPV1Cre::FcεR1γfl/fl mice and in FcεR1α-/- mice into which bone marrow has been transplanted. Finally, increased in vivo circulating levels of IgE following allergen sensitization enhances the responsiveness of FcεR1 to immune complexes in both mouse jugular nodose complex ganglion neurons and human induced pluripotent stem cell-derived nociceptors. CONCLUSIONS: Allergen sensitization triggers a feedforward inflammatory loop between IgE-producing plasma cells, FcεR1-expressing vagal sensory neurons, and TH2 cells, which helps to both initiate and amplify allergic airway inflammation. These data highlight a novel target for reducing allergy, namely, FcεR1γ expressed by nociceptors.

Accum™ Technology: A Novel Conjugable Primer for Onco-Immunotherapy.

Compromised activity is a common impediment for biologics requiring endosome trafficking into target cells. In cancer cells, antibody-drug conjugates (ADCs) are trapped in endosomes or subsequently pumped extracellularly, leading to a reduction in intracellular accumulation. In subsets of dendritic cells (DCs), endosome-engulfed antigens face non-specific proteolysis and collateral damage to epitope immunogenicity before proteasomal processing and subsequent surface presentation. To bypass these shortcomings, we devised Accum™, a conjugable biotechnology harboring cholic acid (ChAc) and a nuclear localization signal (NLS) sequence for endosome escape and prompt nuclear targeting. Combined, these mechanisms culminate in enhanced intracellular accumulation and functionalization of coupled biologics. As proof-of-principle, we have biochemically characterized Accum, demonstrating its adaptability to ADCs or antigens in different cancer settings. Additionally, we have validated that endosome escape and nuclear routing are indispensable for effective intracellular accumulation and guaranteed target cell selectivity. Importantly, we have demonstrated that the unique mechanism of action of Accum translates into enhanced tumor cytotoxicity when coupled to ADCs, and durable therapeutic and prophylactic anti-cancer immunogenicity when coupled to tumor antigens. As more pre-clinical evidence accumulates, the adaptability, unique mechanism of action, and high therapeutic potency of Accum signal a promising transition into clinical investigations in the context of onco-immunotherapy.

Restoring thymic function: Then and now.

Thymic vulnerability, a leading cause of defective immunity, was discovered decades ago. To date, several strategies have been investigated to unveil any immunorestorative capacities they might confer. Studies exploiting castration, transplantation, adoptive cell therapies, hormones/growth factors, and cytokines have demonstrated enhanced in vitro and in vivo thymopoiesis, albeit with clinical restrictions. In this review, we will dissect the thymus on a physiological and pathological level and discuss the pros and cons of several strategies esteemed thymotrophic from a pre-clinical perspective. Finally, we will shed light on interleukin (IL)-21, a pharmacologically-promising cytokine with a significant thymotrophic nature, and elaborate on its potential clinical efficacy and safety in immune-deficient subjects.

Differential effects of γc cytokines on postselection differentiation of CD8 thymocytes.

The primary consequence of positive selection is to render thymocytes responsive to cytokines and chemokines expressed in the thymic medulla. In the present study, our main objective was to discover which cytokines could support the differentiation of positively selected thymocytes. To this end, we have developed an in vitro model suitable for high-throughput analyses of positive selection and CD8 T-cell differentiation. The model involves coculture of TCR(hi)CD5(int)CD69(-) double-positive (DP) thymocytes with peptide-pulsed OP9 cells and γc-cytokines. We report that IL-4, IL-7, and IL-21 have nonredundant effects on positively selected DP thymocytes. IL-7 signaling phosphorylates STAT5 and ERK; induces Foxo1, Klf2, and S1pr1; and supports the differentiation of classic CD8 T cells. IL-4 activates STAT6 and ERK and supports the differentiation of CD8(int)PD-L1(hi)CD44(hi)EOMES(+) innate CD8 T cells. IL-21 is produced by thymic epithelial cells and the IL-21 receptor-α is strongly induced on DP thymocytes undergoing positive selection. IL-21 signaling phosphorylates STAT3 and STAT5, but not ERK, and does not support CD8 T-cell differentiation. However, IL-21 has a unique ability to up-regulate BCL-6, expand DP thymocytes undergoing positive selection, and increase the production of mature T cells. Our data suggest that injection of recombinant IL-21 might enhance thymic output in subjects with age- or disease-related thymic atrophy.

BASOPHILS ACTIVATE PRURICEPTOR-LIKE VAGAL SENSORY NEURONS.

Vagal sensory neurons convey sensations from internal organs along the vagus nerve to the brainstem. Pruriceptors are a subtype of neurons that transmit itch and induce pruritus. Despite extensive research on the molecular mechanisms of itch, studies focusing on pruriceptors in the vagal ganglia still need to be explored. In this study, we characterized vagal pruriceptor neurons by their responsiveness to pruritogens such as lysophosphatidic acid, ß-alanine, chloroquine, and the cytokine oncostatin M. We discovered that lung-resident basophils produce oncostatin M and that its release can be induced by engagement of FcεRIα. Oncostatin M then sensitizes multiple populations of vagal sensory neurons, including Tac1+ and MrgprA3+ neurons in the jugular ganglia. Finally, we observed an increase in oncostatin M release in mice sensitized to the house dust mite Dermatophagoides pteronyssinus or to the fungal allergen Alternaria alternata, highlighting a novel mechanism through which basophils and vagal sensory neurons may communicate during type I hypersensitivity diseases such as allergic asthma.

A1-reprogrammed mesenchymal stromal cells prime potent antitumoral responses.

Mesenchymal stromal cells (MSCs) have been modified via genetic or pharmacological engineering into potent antigen-presenting cells-like capable of priming responding CD8 T cells. In this study, our screening of a variant library of Accum molecule revealed a molecule (A1) capable of eliciting antigen cross-presentation properties in MSCs. A1-reprogrammed MSCs (ARM) exhibited improved soluble antigen uptake and processing. Our comprehensive analysis, encompassing cross-presentation assays and molecular profiling, among other cellular investigations, elucidated A1's impact on endosomal escape, reactive oxygen species production, and cytokine secretion. By evaluating ARM-based cellular vaccine in mouse models of lymphoma and melanoma, we observe significant therapeutic potency, particularly in allogeneic setting and in combination with anti-PD-1 immune checkpoint inhibitor. Overall, this study introduces a strong target for developing an antigen-adaptable vaccination platform, capable of synergizing with immune checkpoint blockers to trigger tumor regression, supporting further investigation of ARMs as an effective and versatile anti-cancer vaccine.

Development and function of innate polyclonal TCRalphabeta+ CD8+ thymocytes.

Innate CD8 T cells are found in mutant mouse models, but whether they are produced in a normal thymus remains controversial. Using the RAG2p-GFP mouse model, we found that ∼10% of TCRαß(+) CD4(-)CD8(+) thymocytes were innate polyclonal T cells (GFP(+)CD44(hi)). Relative to conventional T cells, innate CD8 thymocytes displayed increased cell surface amounts of B7-H1, CD2, CD5, CD38, IL-2Rß, and IL-4Rα and downmodulation of TCRß. Moreover, they overexpressed several transcripts, including T-bet, Id3, Klf2, and, most of all, Eomes. Innate CD8 thymocytes were positively selected, mainly by nonhematopoietic MHCIa(+) cells. They rapidly produced high levels of IFN-γ upon stimulation and readily proliferated in response to IL-2 and IL-4. Furthermore, low numbers of innate CD8 thymocytes were sufficient to help conventional CD8 T cells expand and secrete cytokine following Ag recognition. This helper effect depended on CD44-mediated interactions between innate and conventional CD8 T cells. We concluded that innate TCRαß(+) CD8 T cells represent a sizeable proportion of normal thymocytes whose development and function differ in many ways from those of conventional CD8 T cells.

Inducible IL10(+) suppressor B cells inhibit CNS inflammation and T helper 17 polarization.

Granulocyte-macrophage colony-stimulating factor (GMCSF) and MCP3 (aka CCL7) exert complementary, nonoverlapping, proimmune effects on responsive lymphoid and myeloid cells. We hypothesized that a synthetic cytokine linking GMCSF to MCP3 (hereafter GMME3) as part of a single polypeptide would acquire novel, therapeutically desirable immunomodulatory properties. We demonstrate that GMME3 has enhanced CC-chemokine receptor (CCR)-mediated intracellular Ca(++) mobilization with selective effects on the CD21(hi)CD24(hi) CD1.d(hi) subset of splenic B cells inducing substantial interleukin 10 (IL10) production. We demonstrate that B(GMME3) exert their suppressive effect through an IL10-mediated inhibition of antigen presentation. More importantly, B(GMME3) inhibit the reactivation of encephalomyelitis (EAE)-derived or TGFß/IL6 differentiated Th17 cells by altering their polarization toward a Th1 or Th2 phenotype. The secretion of interferon-γ (IFNγ) and IL4 in turn inhibits IL17 production. The adoptive transfer of B(GMME3), but not IL10(-/-) B(GMME3) cells, to mice symptomatic with experimental autoimmune encephalitis significantly improves their disease score and inhibits lymphoid infiltration into the central nervous system (CNS). We propose that designed CCR modulators such as GMME3, allows for conversion of naive B-cells to a novel suppressor phenotype allowing for the personalized cell therapy of autoimmune ailments.

A dendritic cell population generated by a fusion of GM-CSF and IL-21 induces tumor-antigen-specific immunity.

We have previously shown that the fusion of GM-CSF and IL-21 (GIFT-21) possesses a potent immune stimulatory effect on myeloid cells. In this study, we define the effect of GIFT-21 on naive murine monocytes (GIFT-21 dendritic cells [DCs]), which express increased levels of Gr-1, CD45R, MHC class I, CD80, CD86, and CXCR4 and suppress CD11c and MHC class II. Compared with conventional dendritic cells, GIFT-21 DCs produced substantially more CCL2, IL-6, TNF-α, and IFN-α and induced significantly greater production of IFN-γ by CD8(+) T cells in MHC class I-restricted Ag presentation assays. B16 melanoma and D2F2 Neu breast cancer growth was inhibited in mice treated with Ag-naive GIFT-21 DCs. This effect was lost in CD8(-/-) and CCR2(-/-) mice and when mice were treated with ß(2)-microglobulin-deficient GIFT-21 DCs, indicating that GIFT-21 DCs migrated to and sampled from the tumors to present tumor Ags to CCL2 recruited CD8(+) T cells via MHC class I. We propose that autologous GIFT-21 DCs may serve as a cell therapy platform for the treatment of cancer.

CD146 Defines a Mesenchymal Stromal Cell Subpopulation with Enhanced Suppressive Properties.

Mesenchymal stromal cells (MSCs) are largely known for their immune-suppressive capacity, hence, their common use in the control of unwanted inflammation. However, novel concepts related to their biology, combined with the urgent need to identify MSC subpopulations with enhanced suppressive properties, drive the search for isolation protocols optimized for clinical applications. We show, in this study, that MSCs expressing high CD146 levels exhibit altered surface expression profiles of CD44 and secrete elevated levels of interleukin (IL)-6, amongst other factors. In addition, CD146hi MSCs surpass the polyclonal parental populations in inhibiting alloreactive T cells in vitro, in both a soluble- and cell-contact-dependent manner. Despite the lack of CD146hi MSC-mediated activation of peritoneal macrophages to release the suppressive factor IL-10 in vitro, their administration in animals with graft-versus-host disease alleviates inflammation and leads to 40% survival rate up to 7 weeks post-transplantation. This pronounced inhibitory property is driven by CD146-mediated in situ efferocytosis by myeloid cells. Altogether, this study provides the impetus to adopt an isolation protocol for MSCs based on a CD146 expression profile before their therapeutic use and suggests a major role played by CD146 as a novel "eat-me" signal, capable of enhancing MSC uptake by competent phagocytes.

Recent Advances in Cancer Drug Discovery Through the Use of Phenotypic Reporter Systems, Connectivity Mapping, and Pooled CRISPR Screening.

Multi-omic approaches offer an unprecedented overview of the development, plasticity, and resistance of cancer. However, the translation from anti-cancer compounds identified in vitro to clinically active drugs have a notoriously low success rate. Here, we review how technical advances in cell culture, robotics, computational biology, and development of reporter systems have transformed drug discovery, enabling screening approaches tailored to clinically relevant functional readouts (e.g., bypassing drug resistance). Illustrating with selected examples of "success stories," we describe the process of phenotype-based high-throughput drug screening to target malignant cells or the immune system. Second, we describe computational approaches that link transcriptomic profiling of cancers with existing pharmaceutical compounds to accelerate drug repurposing. Finally, we review how CRISPR-based screening can be applied for the discovery of mechanisms of drug resistance and sensitization. Overall, we explore how the complementary strengths of each of these approaches allow them to transform the paradigm of pre-clinical drug development.

Teasing Out the Interplay Between Natural Killer Cells and Nociceptor Neurons.

Somatosensory neurons have evolved to detect noxious stimuli and activate defensive reflexes. By sharing means of communication, nociceptor neurons also tune host defenses by controlling the activity of the immune system. The communication between these systems is mostly adaptive, helping to protect homeostasis, it can also lead to, or promote, the onset of chronic diseases. Both systems co-evolved to allow for such local interaction, as found in primary and secondary lymphoid tissues and mucosa. Recent studies have demonstrated that nociceptors directly detect and respond to foreign antigens, immune cell-derived cytokines, and microbes. Nociceptor activation not only results in pain hypersensitivity and itching, but lowers the nociceptor firing threshold, leading to the local release of neuropeptides. The peptides that are produced by, and released from, the peripheral terminals of nociceptors can block the chemotaxis and polarization of lymphocytes, controlling the localization, duration, and type of inflammation. Recent evidence shows that sensory neurons interact with innate immune cells via cell-cell contact, for example, engaging group 2D (NKG2D) receptors on natural killer (NK) cells. Given that NK cells express the cognate receptors for various nociceptor-produced mediators, it is conceivable that nociceptors use neuropeptides to control the activity of NK cells. Here, we devise a co-culture method to study nociceptor neuron-NK cell interactions in a dish. Using this approach, we found that lumbar nociceptor neurons decrease NK cell cytokine expression. Overall, such a reductionist method could be useful to study how tumor-innervating neurons control the anticancer function of NK cells and how NK cells control the elimination of injured neurons.

Humoral Immunity to Allogeneic Immunoproteasome-Expressing Mesenchymal Stromal Cells Requires Efferocytosis by Endogenous Phagocytes.

The extensive use of mesenchymal stromal cells (MSCs) over the last decade has revolutionized modern medicine. From the delivery of pharmacological proteins to regenerative medicine and immune modulation, these cells have proven to be highly pleiotropic and responsive to their surrounding environment. Nevertheless, their role in promoting inflammation has been fairly limited by the questionable use of interferon-gamma, as this approach has also been proven to enhance the cells' immune-suppressive abilities. Alternatively, we have previously shown that de novo expression of the immunoproteasome (IPr) complex instills potent antigen cross-presentation capabilities in MSCs. Interestingly, these cells were found to express the major histocompatibility class (MHC) II protein, which prompted us to investigate their ability to stimulate humoral immunity. Using a series of in vivo studies, we found that administration of allogeneic ovalbumin (OVA)-pulsed MSC-IPr cells elicits a moderate antibody titer, which was further enhanced by the combined use of pro-inflammatory cytokines. The generated antibodies were functional as they blocked CD4 T-cell activation following their co-culture with OVA-pulsed MSC-IPr and mitigated E.G7 tumor growth in vivo. The therapeutic potency of MSC-IPr was, however, dependent on efferocytosis, as phagocyte depletion prior to vaccination abrogated MSC-IPr-induced humoral responses while promoting their survival in the host. In contrast, antibody-mediated neutralization of CD47, a potent "do not eat me signal", enhanced antibody titer levels. These observations highlight the major role played by myeloid cells in supporting antibody production by MSC-IPr and suggest that the immune outcome is dictated by a net balance between efferocytosis-stimulating and -inhibiting signals.

The CIt protocol: A blueprint to potentiate the immunogenicity of immunoproteasome-reprogrammed mesenchymal stromal cells.

Immunoproteasome-reprogrammed mesenchymal stromal cells (IRMs) can surpass dendritic cells at eliciting tumor-specific immunity. However, the current IRM vaccination regimen remains clinically unsuitable due to the relatively high dose of IRMs needed. Since the administration of a lower IRM dose triggers a feeble anti-tumoral response, we aimed to combine this vaccination regimen with different modalities to fine-tune the potency of the vaccine. In a nutshell, we found that the co-administration of IRMs and interleukin-12 accentuates the anti-tumoral response, whereas the cross-presentation potency of IRMs is enhanced via intracellular succinate build-up, delayed endosomal maturation, and increased endosome-to-cytosol plasticity. Stimulating phagocyte-mediated cancer efferocytosis by blocking the CD47-SIRPα axis was also found to enhance IRM vaccine outcomes. Upon designing a single protocol combining the abovementioned strategies, 60% of treated animals exhibited a complete response. Altogether, this is the first IRM-based vaccination study, optimized to simultaneously target three vaccine-related pitfalls: T-cell response, antigen cross-presentation, and cancer phagocytosis.