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.

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.

MFG-E8 Reduces Aortic Intimal Proliferation in a Murine Model of Transplant Vasculopathy.

Transplant vasculopathy is characterized by endothelial apoptosis, which modulates the local microenvironment. Milk fat globule epidermal growth factor 8 (MFG-E8), which is released by apoptotic endothelial cells, limits tissue damage and inflammation by promoting anti-inflammatory macrophages. We aimed to study its role in transplant vasculopathy using the murine aortic allotransplantation model. BALB/c mice were transplanted with fully mismatched aortic transplants from MFG-E8 knockout (KO) or wild type (WT) C57BL/6J mice. Thereafter, mice received MFG-E8 (or vehicle) injections for 9 weeks prior to histopathological analysis of allografts for intimal proliferation (hematoxylin and eosin staining) and leukocyte infiltration assessment (immunofluorescence). Phenotypes of blood leukocytes and humoral responses were also evaluated (flow cytometry and ELISA). Mice receiving MFG-E8 KO aortas without MFG-E8 injections had the most severe intimal proliferation (p < 0.001). Administration of MFG-E8 decreased intimal proliferation, especially in mice receiving MFG-E8 KO aortas. Administration of MFG-E8 also increased the proportion of anti-inflammatory macrophages among graft-infiltrating macrophages (p = 0.003) and decreased systemic CD4+ and CD8+ T-cell activation (p < 0.001). An increase in regulatory T cells occurred in both groups of mice receiving WT aortas (p < 0.01). Thus, the analarmin MFG-E8 appears to be an important protein for reducing intimal proliferation in this murine model of transplant vasculopathy. MFG-E8 effects are associated with intra-allograft macrophage reprogramming and systemic T-cell activation dampening.

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.

Exploring cannabidiol effects on inflammatory markers in individuals with cocaine use disorder: a randomized controlled trial.

Cocaine use disorder (CUD) is a major public health issue associated with physical, social, and psychological problems. Excessive and repeated cocaine use induces oxidative stress leading to a systemic inflammatory response. Cannabidiol (CBD) has gained substantial interest for its anti-inflammatory properties, safety, and tolerability profile. However, CBD anti-inflammatory properties have yet to be confirmed in humans. This exploratory study is based on a single-site randomized controlled trial that enrolled participants with CUD between 18 and 65 years, randomized (1:1) to daily receive either CBD (800 mg) or placebo for 92 days. The trial was divided into a 10-day detoxification (phase I) followed by a 12-week outpatient follow-up (phase II). Blood samples were collected from 48 participants at baseline, day 8, week 4, and week 12 and were analyzed to determine monocytes and lymphocytes phenotypes, and concentrations of various inflammatory markers such as cytokines. We used generalized estimating equations to detect group differences. Participants treated with CBD had lower levels of interleukin-6 (p = 0.017), vascular endothelial growth factor (p = 0.032), intermediate monocytes CD14+CD16+ (p = 0.024), and natural killer CD56negCD16hi (p = 0.000) compared with participants receiving placebo. CD25+CD4+T cells were higher in the CBD group (p = 0.007). No significant group difference was observed for B lymphocytes. This study suggests that CBD may exert anti-inflammatory effects in individuals with CUD.

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.

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.

Peripheral and local predictive immune signatures identified in a phase II trial of ipilimumab with carboplatin/paclitaxel in unresectable stage III or stage IV melanoma.

BACKGROUND: Checkpoint blockade with ipilimumab provides long-term survival to a significant proportion of patients with metastatic melanoma. New approaches to increase survival and to predict which patients will benefit from treatment are needed. This phase II trial combined ipilimumab with carboplatin/paclitaxel (CP) to assess its safety, efficacy, and to search for peripheral and tumor-based predictive biomarkers. METHODS: Thirty patients with untreated unresectable/metastatic melanoma were treated with ipilimumab and CP. Adverse events (AEs) were monitored and response to treatment was evaluated. Tumor tissue and peripheral blood were collected at specified time points to characterize tumor immune markers by immunohistochemistry and systemic immune activity by multiplex assays and flow cytometry. RESULTS: Eighty three percent of patients received all 5 cycles of CP and 93% completed ipilimumab induction. Serious AEs occurred in 13% of patients, and no treatment-related deaths were observed. Best Overall Response Rate (BORR) and Disease Control Rate (DCR) were 27 and 57%, respectively. Median overall survival was 16.2 months. Response to treatment was positively correlated with a higher tumor CD3+ infiltrate (immune score) at baseline. NRAS and BRAF mutations were less frequent in patients who experienced clinical benefit. Assessment of peripheral blood revealed that non-responders had elevated baseline levels of CXCL8 and CCL4, and a higher proportion of circulating late differentiated B cells. Pre-existing high levels of chemokines (CCL3, CCL4 and CXCL8) and advanced B cell differentiation were strongly associated with worse patient overall survival. Elevated proportions of circulating CD8+/PD-1+ T cells during treatment were associated with worse survival. CONCLUSIONS: The combination of ipilimumab and CP was well tolerated and revealed novel characteristics associated with patients likely to benefit from treatment. A pre-existing systemic inflammatory state characterized by elevation of selected chemokines and advanced B cell differentiation, was strongly associated with poor patient outcomes, revealing potential predictive circulating biomarkers. TRIAL REGISTRATION: Clinicaltrials.gov , NCT01676649 , registered on August 29, 2012.

The non-classical MAP kinase ERK3 controls T cell activation.

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

Hoxb4 overexpression in CD4 memory phenotype T cells increases the central memory population upon homeostatic proliferation.

Memory T cell populations allow a rapid immune response to pathogens that have been previously encountered and thus form the basis of success in vaccinations. However, the molecular pathways underlying the development and maintenance of these cells are only starting to be unveiled. Memory T cells have the capacity to self renew as do hematopoietic stem cells, and overlapping gene expression profiles suggested that these cells might use the same self-renewal pathways. The transcription factor Hoxb4 has been shown to promote self-renewal divisions of hematopoietic stem cells resulting in an expansion of these cells. In this study we investigated whether overexpression of Hoxb4 could provide an advantage to CD4 memory phenotype T cells in engrafting the niche of T cell deficient mice following adoptive transfer. Competitive transplantation experiments demonstrated that CD4 memory phenotype T cells derived from mice transgenic for Hoxb4 contributed overall less to the repopulation of the lymphoid organs than wild type CD4 memory phenotype T cells after two months. These proportions were relatively maintained following serial transplantation in secondary and tertiary mice. Interestingly, a significantly higher percentage of the Hoxb4 CD4 memory phenotype T cell population expressed the CD62L and Ly6C surface markers, characteristic for central memory T cells, after homeostatic proliferation. Thus Hoxb4 favours the maintenance and increase of the CD4 central memory phenotype T cell population. These cells are more stem cell like and might eventually lead to an advantage of Hoxb4 T cells after subjecting the cells to additional rounds of proliferation.

Distinct tryptophan catabolism and Th17/Treg balance in HIV progressors and elite controllers.

Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) by indoleamine 2,3-dioxygenase (IDO) was previously linked to Th17/Treg differentiation and immune activation. Here we examined Trp catabolism and its impact on Th17/Treg balance in uninfected healthy subjects (HS) and a large cohort of HIV-infected patients with different clinical outcomes: ART-naïve, Successfully Treated (ST), and elite controllers (EC). In ART-naïve patients, increased IDO activity/expression, together with elevated levels of TNF-α and sCD40L, were associated with Treg expansion and an altered Th17/Treg balance. These alterations were normalized under ART. In contrast, Trp 2,3-dioxegenase (TDO) expression was dramatically lower in EC when compared to all other groups. Interestingly, EC displayed a distinctive Trp metabolism characterized by low Trp plasma levels similar to ART-naïve patients without accumulating immunosuppressive Kyn levels which was accompanied by a preserved Th17/Treg balance. These results suggest a distinctive Trp catabolism and Th17/Treg balance in HIV progressors and EC. Thus, IDO-induced immune-metabolism may be considered as a new inflammation-related marker for HIV-1 disease progression.

Notch signaling regulates PD-1 expression during CD8(+) T-cell activation.

Programmed cell death 1 (PD-1) is an inhibitory receptor involved in T-cell activation, tolerance and exhaustion. Little is known on how the expression of PD-1 is controlled during T-cell activation. Recent studies demonstrated that NFATc1 and IRF9 regulate Pdcd1 (PD-1) transcription and that T-bet acts as a transcriptional repressor. In this study, we have investigated the role of the Notch signaling pathway in PD-1 regulation. Using specific inhibitors of the Notch signaling pathway, we showed decreased PD-1 expression and inhibition of Pdcd1 transcription by activated CD8(+) T cells. Chromatin immunoprecipitation further showed occupancy of the Pdcd1 promoter with RBPJk and Notch1 intracellular domain at RBPJk-binding sites. Our results identify the Notch signaling pathway as an important regulator of PD-1 expression by activated CD8(+) T cells.

Expression of heme oxygenase-1 in neural stem/progenitor cells as a potential mechanism to evade host immune response.

Besides their therapeutic benefit as cell source, neural stem/progenitor cells (NSPCs) exhibit immunosuppressive properties of great interest for modulating immune response in the central nervous system. To decipher the mechanisms of NSPC-mediated immunosuppression, activated T cells were exposed to NSPCs isolated from fetal rat brains. Analyses revealed that NSPCs inhibited T-cell proliferation and interferon-gamma production in a dose-dependent manner. A higher proportion of helper T cells (CD4+ T cells) was found in the presence of NSPCs, but analyses of FoxP3 population indicated that T-cell suppression was not secondary to an induction of suppressive regulatory T cells (FoxP3+ CD4+ CD25+). Conversely, induction of the high affinity interleukin-2 (IL-2) receptor (CD25) and the inability of IL-2 to rescue T-cell proliferation suggest that NSPCs display immunosuppressive activity without affecting T-cell activation. Cultures in Transwell chambers or addition of NSPC-conditioned medium to activated T cells indicated that part of the suppressive activity was not contact dependent. We therefore searched for soluble factors that mediate NSPC immunosuppression. We found that NSPCs express several immunosuppressive molecules, but the ability of these cells to inhibit T-cell proliferation was only counteracted by heme oxygenase (HO) inhibitors in association or not with nitric oxide synthase inhibitors. Taken together, our findings highlight a dynamic crosstalk between NSPCs and T lymphocytes and provide the first evidence of an implication of HO-1 in mediating the immunosuppressive effects of the NSPCs.