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Mesenchymal stromal cells (MSCs) have recently emerged as an interesting therapeutic approach for patients with progressive systemic sclerosis (SSc), a rare and life-threatening orphan autoimmune disease. Whereas MSC immunomodulatory potential is considered as a central mechanism for their clinical benefit, very few data are available on the impact of MSCs on immune cell subsets in vivo. In the current extended study of a phase I/II clinical trial exploring the injection of a single dose of allogeneic bone marrow-MSCs (alloBM-MSCs) in patients with severe SSc (NCT02213705), we performed a longitudinal in-depth characterization of circulating immune cells in 19 MSC-treated patients, including 14 responders and 5 non-responders. By a combination of flow cytometry and transcriptomic analyses, we highlighted an increase in circulating CD24hiCD27posCD38lo/neg memory B cells, the main IL-10-producing regulatory B cell (Breg) subset, and an upregulation of IL10 expression in ex-vivo purified B cells, specifically in responder patients, early after the alloBM-MSC infusion. In addition, a deeper alteration of the B-cell compartment before alloBM-MSC treatment, including a higher expression of profibrotic cytokines IL6 and TGFβ by sorted B cells was associated with a non-responder clinical status. Finally, BM-MSCs were able to directly upregulate IL-10 production in activated B cells in vitro. These data suggest that cytokine-producing B cells, in particular Breg, are pivotal effectors of BM-MSC therapeutic activity in SSc. Their quantification as activity biomarkers in MSC potency assays and patient selection criteria may be considered to reach optimal clinical benefit when designing MSC-based clinical trials.

Supplementary Figure 2 from Functional Alteration of the Lymphoma Stromal Cell Niche by the Cytokine Context: Role of Indoleamine-2,3 Dioxygenase

Supplementary Figure 1 from Functional Alteration of the Lymphoma Stromal Cell Niche by the Cytokine Context: Role of Indoleamine-2,3 Dioxygenase

Supplementary Figure 3 from Functional Alteration of the Lymphoma Stromal Cell Niche by the Cytokine Context: Role of Indoleamine-2,3 Dioxygenase

Supplementary Figure Legends 1-3 from Functional Alteration of the Lymphoma Stromal Cell Niche by the Cytokine Context: Role of Indoleamine-2,3 Dioxygenase

Supplementary Table 1 from Functional Alteration of the Lymphoma Stromal Cell Niche by the Cytokine Context: Role of Indoleamine-2,3 Dioxygenase

Human mesenchymal stem cells (MSC) strongly repress activated T-cell proliferation through the production of a complex set of soluble factors, including the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which is induced by IFN-γ. Conversely, MSCs support survival of follicular lymphoma (FL) B cells, in particular after exposure to tumor necrosis factor-α (TNF) and lymphotoxin-α1β2 (LT). The role of MSCs on normal and malignant B-cell growth in steady-state and inflammatory conditions remains to be fully explored. We show here that resting MSCs sustain activated normal B-cell proliferation and survival, whereas IFN-γ–conditioned MSCs mediate IDO–dependent B-cell growth arrest and apoptosis. IFN-γ, TNF, and LT are significantly overexpressed by the microenvironment of invaded FL-lymph nodes, but their relative expression patterns are highly heterogeneous between samples. In vitro, IFN-γ abrogates the B-cell supportive phenotype induced by TNF and LT on MSCs. Moreover, IFN-γ overrules the growth promoting effect of MSCs on primary purified FL B cells. Altogether, these results underline the crucial role of the cytokine context in the local crosstalk between malignant cells and their microenvironment and provide new insights into our knowledge of the FL cell niche that emerges as a new promising target for innovative therapeutic strategies. [Cancer Res 2009;69(7):3228–37]

International audience; The therapeutic potential of culture-adapted adipose-derived stromal cells (ASCs) is largely related to their production of immunosuppressive factors that are inducible in vitro by priming with inflammatory stimuli, in particular tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). In vivo, obesity is associated with chronic inflammation of white adipose tissue, including accumulation of neutrophils, infiltration by IFNγ/TNFα-producing immune cells, and ASC dysfunction. In the current study, we identified in obese patients a simultaneous upregulation of CD40Lin the adipose tissue stroma vascular fraction (AT-SVF), correlated with the Th1 gene signature, and an overexpression of CD40 by native ASCs. Moreover, activated CD4+ T cells upregulated CD40 on culture-expanded ASCs and triggered their production of IL-8 in a CD40L-dependent manner, leading to an increased capacity to recruit neutrophils. Finally, activation of ASCs by sCD40L or CD40L-expressing CD4+ T cells relies on both canonical and non-canonical NF-κB pathways, and IL-8 was found to be coregulated with NF-κB family members in AT-SVF. These data identify the CD40-CD40L axis as a priming mechanism of ASCs, able to modulate their cross talk with neutrophils in an inflammatory context, and their functional capacity for therapeutic applications.

The therapeutic potential of culture-adapted adipose-derived stromal cells (ASCs) is largely related to their production of immunosuppressive factors that are inducible in vitro by priming with inflammatory stimuli, in particular tumor necrosis factor-α (TNFα) and interferon-γ (IFNγ). In vivo, obesity is associated with chronic inflammation of white adipose tissue, including accumulation of neutrophils, infiltration by IFNγ/TNFα-producing immune cells, and ASC dysfunction. In the current study, we identified in obese patients a simultaneous upregulation of CD40Lin the adipose tissue stroma vascular fraction (AT-SVF), correlated with the Th1 gene signature, and an overexpression of CD40 by native ASCs. Moreover, activated CD4

Acute respiratory distress syndrome (ARDS) is the main complication of COVID-19, requiring admission to Intensive Care Unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS differs from other causes of ARDS remains unknown. To address this question, we build 3 cohorts of patients categorize in COVID-19negARDSpos, COVID-19posARDSpos, and COVID-19posARDSneg, and compare their immune landscape analyze by high-dimensional mass cytometry on peripheral blood. A cell signature associating S100A9/calprotectin-producing CD169pos monocytes, plasmablasts, and Th1 cells is found in COVID-19posARDSpos, unlike COVID-19negARDSpos patients. Moreover, this signature is essentially share with COVID-19posARDSneg patients, suggesting that severe COVID-19 patients, whatever they experience or not ARDS, display similar immune profiles. We show an increase in CD14posHLA-DRlow and CD14lowCD16pos monocytes correlate to the occurrence of adverse events during ICU stay. We demonstrate that COVID-19-associated ARDS display a specific immune profile, and might benefit from personalize therapy in addition to standard ARDS management., Graphical Abstract, Roussel et al. characterize the immune profile of COVID-19+ and COVID-19- patients, both presenting an acute respiratory distress syndrome (ARDS) and COVID-19+ without ARDS. They identify a COVID-19 signature associating CD169+S100A9+ monocytes, plasmablasts, and Th1 cells. CD14+HLA-DRlo and CD14loCD16+ monocytes increase during the ICU stay correlate with unfavorable clinical course.

The immunomodulatory drug lenalidomide is used in patients with follicular lymphoma (FL) with the aim of stimulating T-cell antitumor immune response. However, little is known about the effects of lenalidomide on T-cell biology in vivo in patients with FL. We thus undertook an extensive longitudinal immunologic study, including phenotypic, transcriptomic, and functional analyses, on 44 first-line and 27 relapsed/refractory patients enrolled in the GALEN trial (Obinutuzumab Combined With Lenalidomide for Relapsed or Refractory Follicular B-Cell Lymphoma) to test the efficacy of lenalidomide and obinutuzumab combination in patients with FL. Lenalidomide rapidly and transiently induced an activated T-cell phenotype, including HLA-DR, Tim-3, CD137, and programmed cell death protein 1 (PD-1) upregulation. Furthermore, sequential RNA-sequencing of sorted PD-1+ and PD-1– T-cell subsets revealed that lenalidomide triggered a strong enrichment for several gene signatures related to effector memory T-cell features, including proliferation, antigen receptor signaling, and immune synapse restoration; all were validated at the phenotypic level and with ex vivo functional assays. Correlative analyses pinpointed a negative clinical impact of high effector T-cell and regulatory T-cell percentages before and during treatment. Our findings bring new insight in lenalidomide mechanisms of action at work in vivo and will fuel a new rationale for the design of combination therapies.

Acute respiratory distress syndrome (ARDS) is the main complication of COVID-19, requiring admission to Intensive Care Unit (ICU). Despite recent immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS specifically differs from other causes of ARDS remains unknown, To address this question, we built 3 cohorts of patients categorized in COVID-19negARDSpos, COVID-19posARDSpos, and COVID-19posARDSneg, and compared their immune landscape analyzed by high-dimensional mass cytometry on peripheral blood followed by artificial intelligence analysis. A cell signature associating S100A9/calprotectin-producing CD169pos monocytes, plasmablasts, and Th1 cells was specifically found in COVID-19posARDSpos, unlike COVID-19negARDSpos patients. Moreover, this signature was shared by COVID-19posARDSneg patients, suggesting severe COVID-19 patients, whatever they experienced or not ARDS, displayed similar immune dysfunctions. We also showed an increase in CD14posHLA-DRlow and CD14lowCD16pos monocytes correlated to the occurrence of adverse events during ICU stay. Our study demonstrates that COVID-19-associated ARDS display a specific immune profile, and might benefit from personalized therapy in addition to standard ARDS management.One Sentence SummaryCOVID-19-associated ARDS is biologically distinct from other causes of ARDS.

Acute respiratory distress syndrome (ARDS) is the main complication of COVID-19, requiring admission to Intensive Care Unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS specifically differs from other causes of ARDS remains unknown. To address this crucial question, we built 3 cohorts of patients categorized in COVID-19neg ARDSpos, COVID-19pos ARDSpos, and COVID-19pos ARDSneg, and compared their immune landscape analyzed by high-dimensional mass cytometry on peripheral blood followed by artificial intelligence analysis. A cell signature associating S100A9/calprotectin-producing CD169pos monocytes, plasmablasts, and Th1 cells was specifically found in COVID-19pos ARDSpos, unlike COVID-19neg ARDSpos patients. Moreover, this signature was essentially shared with COVID-19pos ARDSneg patients, suggesting that severe COVID-19 patients, whatever they experienced or not ARDS, display similar immune profiles. We also showed an increase in CD14pos HLA-DRlow and CD14low CD16pos monocytes correlated to the occurrence of adverse events during ICU stay. Our study demonstrates that COVID-19-associated ARDS display a specific immune profile, and might benefit from personalized therapy in addition to standard ARDS management. Funding: This work was supported by the University hospital of Rennes, CFTR² (COVID-19 Fast Track Recherche Rennes) grant (to F.R.)and by the Fondation pour la Recherche Medicale (FRM) and the Agence Nationale de la Recherche (ANR), Flash Covid-19 joint grant (HARMONICOV to M.Cog.) Conflict of Interest: J.Fer., F.R., S.L.G., J.D., M.Le., M.G., N.B., C.V., M.La., I.B., M.Cor., S.L., J.Feu., M. Cog. declare no competing interest. M.R., S.C., V.K.T., J.M.T., and K.T. are the inventors of a patent EP 20305642.9 “A method for early detection of propensity to severe clinical manifestations Methods” submitted June 11th 2020 under University hospital of Rennes and Scailyte AG names. Ethical Approval: The study design was approved by our ethic committee (CHU Rennes, n°35RC20_9795_HARMONICOV, ClinicalTrials.gov Identifier: NCT04373200) and informed consent was obtained from patients in accordance with the Declaration of Helsinki.

Clinical-grade mesenchymal stromal cells (MSCs) can be expanded from bone marrow and adipose tissue to treat inflammatory diseases and degenerative disorders. However, the influence of their tissue of origin on their functional properties, including their immunosuppressive activity, remains unsolved. In this study, we produced paired bone marrow-derived mesenchymal stromal cell (BM-MSC) and adipose-derived stromal cell (ASC) batches from 14 healthy donors. We then compared them using transcriptomic, phenotypic, and functional analyses and validated our results on purified native MSCs to infer which differences were really endowed by tissue of origin. Cultured MSCs segregated together owing to their tissue of origin based on their gene expression profile analyzed using differential expression and weighted gene coexpression network analysis. This translated into distinct immune-related gene signatures, phenotypes, and functional cell interactions. Importantly, sorted native BM-MSCs and ASCs essentially displayed the same distinctive patterns than their in vitro-expanded counterparts. As a whole, ASCs exhibited an immune profile consistent with a stronger inhibition of immune response and a lower immunogenicity, supporting the use of adipose tissue as a valuable source for clinical applications.

Owing to their immunosuppressive properties, mesenchymal stromal cells (MSCs) obtained from bone marrow (BM-MSCs) or adipose tissue (ASCs) are considered a promising tool for cell therapy. However, important issues should be considered to ensure the reproducible production of efficient and safe clinical-grade MSCs. In particular, high expansion rate, associated with progressive senescence, was recently proposed as one of the parameters that could alter MSC functionality. In this study, we directly address the consequences of replicative senescence on BM-MSC and ASC immunomodulatory properties. We demonstrate that MSCs produced according to GMP procedures inhibit less efficiently T-cell, but not Natural Killer (NK)- and B-cell, proliferation after reaching senescence. Senescence-related loss-of-function is associated with a decreased indoleamine 2,3-dioxygenase (IDO) activity in response to inflammatory stimuli. In particular, although STAT-1-dependent IDO expression is transcriptionally induced at a similar level in senescent and nonsenescent MSCs, IDO protein is specifically degraded by the proteasome in senescent ASCs and BM-MSCs, a process that could be reversed by the MG132 proteasome inhibitor. These data encourage the use of appropriate quality controls focusing on immunosuppressive mechanisms before translating clinical-grade MSCs in the clinic.

The immunostimulatory properties of lenalidomide have been mostly described in vitro while in vivo studies performed in multiple myeloma or chronic lymphocytic leukemia have reported a poorly characterized T-cell activation. A better understanding of lenalidomide kinetics and mechanisms of action is mandatory to optimize its combination with other immunotherapeutic agents in particular for the treatment of non Hodgkin lymphomas. We undertook a thorough immune monitoring of patients enrolled in the French multicenter clinical trial GALEN (ClinicalTrials.gov: NCT01582776) addressing the tolerance and efficacy of the association of lenalidomide and obinutuzumab, a glycoengineered type II anti-CD20 monoclonal antibody, in relapsed/refractory B-cell lymphomas. A 1-week interval between the start of lenalidomide and the first infusion of obinutuzumab was planned, allowing an assessment of the effect of lenalidomide alone on immune-related parameters separately from the combinatorial therapy. Serial blood samples were collected in 44 patients (16 DLBCL and 28 FL) to investigate T, B, NK, and myeloid subsets. In addition, in vitro functional assays were designed to address T cell functional features (proliferation, immune synapse, activity of regulatory T cells (Treg)). In the context of the association with obinutuzumab, we first checked that CD20 expression was not affected on circulating malignant and normal B cells (p=0.43 and 0.8; respectively). Interestingly, upon 1 week of lenalidomide treatment, normal B cells, unlike malignant B cells, upregulated MHC class II (p We herein report for the first time early in vivo T cell activation by lenalidomide in relapse FL/DLBCL through a detailed phenotypic analysis strengthened by innovative functional assays. The study of T cells heterogeneity at the transcriptomic level is underway and the correlation of these immunomodulatory properties with clinical data is also currently being addressed. Our results will help build new and more relevant lenalidomide-based immunotherapeutic approaches. (This study was supported by research grants from Celgene and Roche companies) Disclosures Menard: Celgene: Consultancy; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Lamy: Roche: Consultancy, Honoraria. Morschhauser: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Servier: Consultancy; Gilead: Consultancy. Tarte: Celgene: Consultancy, Research Funding; Novimmune: Research Funding; Roche: Consultancy.

International audience; BACKGROUND: Severe sepsis results in a sustained deleterious immune dysregulation. Indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme of tryptophan catabolism, plays a pivotal role in immune tolerance and is induced during various inflammatory conditions. METHODS: Plasma samples obtained from patients with septic shock (n = 38), severe sepsis (n = 35), or sepsis (n = 10) and from healthy donors (n = 26) were analyzed for IDO activity by high-performance liquid chromatography. Lymphocyte, monocyte, and regulatory T cell counts as well as monocytic human leukocyte antigen DR (HLA-DR) expression were quantified by flow cytometry. Peripheral blood mononuclear cells and purified CD14(+) and CD14(-) fractions were assayed in vitro for spontaneous and inducible IDO expression and activity. RESULTS: IDO activity gradually increased according to sepsis severity, and septic patients who died had higher IDO activity on admission than did survivors (P = .013). Monocytes were a major source of active IDO in normal peripheral blood. The percentage and absolute number of circulating CD14(+) cells were increased in septic patients, and their monocytes remained fully able to produce functional IDO after NF-kappaB-independent interferon gamma stimulation but not through NF-kappaB-dependent Toll-like receptor engagement. CONCLUSIONS: IDO activity is increased during severe sepsis and septic shock and is associated with mortality. IDO production could be used to better characterize monocyte reprogramming in sepsis.

Human mesenchymal stem cells (MSC) strongly repress activated T-cell proliferation through the production of a complex set of soluble factors, including the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO), which is induced by IFN-γ. Conversely, MSCs support survival of follicular lymphoma (FL) B cells, in particular after exposure to tumor necrosis factor-α (TNF) and lymphotoxin-α1β2 (LT). The role of MSCs on normal and malignant B-cell growth in steady-state and inflammatory conditions remains to be fully explored. We show here that resting MSCs sustain activated normal B-cell proliferation and survival, whereas IFN-γ–conditioned MSCs mediate IDO–dependent B-cell growth arrest and apoptosis. IFN-γ, TNF, and LT are significantly overexpressed by the microenvironment of invaded FL-lymph nodes, but their relative expression patterns are highly heterogeneous between samples. In vitro, IFN-γ abrogates the B-cell supportive phenotype induced by TNF and LT on MSCs. Moreover, IFN-γ overrules the growth promoting effect of MSCs on primary purified FL B cells. Altogether, these results underline the crucial role of the cytokine context in the local crosstalk between malignant cells and their microenvironment and provide new insights into our knowledge of the FL cell niche that emerges as a new promising target for innovative therapeutic strategies. [Cancer Res 2009;69(7):3228–37]

Loss of surface human leukocyte antigen-DR (HLA-DR) on monocytes is a major factor of immunosuppression in sepsis. Granulocyte macrophage-colony stimulating factor (GM-CSF) up-regulates HLA-DR expression on monocytes via the GM-CSF receptor (GM-CSFr) through a transcriptional mechanism involving the class II transactivator factor (CIITA). We investigated monocyte GM-CSFr expression and its relationship with HLA-DR in septic patients.Prospective clinical experimental study.University hospital intensive care unit and research facility.Septic patients with and without septic shock, control patients.Flow cytometry and real-time quantitative reverse polymerase chain reaction were used to characterize GM-CSFr expression and transcription in septic patients and in ex vivo stimulated healthy monocytes.We showed an early GM-CSFr down-modulation in patients with septic shock compared with those without septic shock and controls. A persistent low GM-CSFr expression was observed in patients who acquired secondary infections or in those who died, and this persistent defect correlated with severity scores. We demonstrated that GM-CSFr down-modulation occurs at a posttranscriptional level since we observed no alteration in GM-CSFr transcription in monocytes isolated from septic patients. Furthermore, we demonstrated that GM-CSFr expression levels on monocytes correlated not only with HLA-DR expression and transcription levels but also with RNA levels of its main transcriptional factor CIITA. Because we previously showed in septic patients a relationship between high cortisol plasma level and low monocyte HLA-DR expression, we investigated the effects of glucocorticoids on monocyte GM-CSFr expression and observed a similar posttranscriptional down-modulation of GM-CSFr by steroids. However, the in vivo putative role of steroids in HLA-DR down-regulation via GM-CSFr down-modulation needs further investigation.Monocyte GM-CSFr down-modulation occurred in septic shock, was associated with severity, and might be either another manifestation of monocyte deactivation linked to sepsis or an additional mechanism participating in immunosuppression.

MSC have been demonstrated to display non-HLA restricted immunosuppressive capacities in vitro and in vivo. Encouraging clinical results have been obtained using MSC infusion for the treatment of steroid-resistant acute graft-versus-host disease (aGVHD). However, very few data are available concerning the capacity of MSC to respond to inflammatory factors that are highly expressed during aGVHD or the influence of these factors on MSC outcome and immunological properties. In particular, the modulation, by environmental factors, of the production of the immunosuppressive enzyme indoleamine-2,3 dioxygenase (IDO) by MSC remains unexplored. The aim of our study was thus to evaluate the influence of the main inflammatory factors produced during GVHD on immunological properties of human clinical-grade MSC. We first confirmed that MSC expressed functional INFγR1 and TNFR1 since IFNγ and TNFα induced a broad set of genes, including those coding for the inflammatory chemokines CXCL9 and CXCL10. During GVHD, a disruption of gastro-intestinal tract integrity occurs and is associated with the release of various microbial compounds that are sensed by Toll Like Receptors (TLR). Using quantitative RT-PCR, we performed an exhaustive analysis of TLR expression on MSC. Whereas TLR2, TLR5, and TLR9 were essentially absent, TLR3 and TLR4, were highly expressed on human clinical-grade MSC. Accordingly, stimulation of MSC by poly(I:C) or LPS induced expression of CXCL9 and CXCL10. Lastly, we focused our work on CD40/CD40L interaction since a high proportion of CD4posT lymphocytes is activated during GVHD and expresses CD40L. We demonstrated that CD40 was present at the beginning of clinical-grade MSC culture and was significantly lost, both at RNA and protein level, after several cell passages. We also provided some evidence that CD40 was uniformly and highly expressed in vivo on CD45negCD14negCD73pos bone marrow cells that were previously shown to be highly enriched for MSC. Importantly, stimulation of MSC by trimeric CD40L was associated with a strong activation of NF-κB signalling pathway and the induction of IL8 gene expression. We then assessed the functional consequences of MSC stimulation by inflammatory factors on their immunological properties. IFNγ and TNFα, unlike TLR ligands and CD40L, induced the expression of relevant immunological markers such as HLA-I, HLA-II, CD54, CD106 and CD40. CD80 and CD86 were not inducible. Despite these phenotypic modifications, MSC did not become immunogenic and maintained their immunosuppressive properties. However, TNFα decreased and IFNγ increased this inhibitory effect. Interestingly, whereas IDO RNA was induced by TNFα and TLR ligands, IFNγ was the only inflammatory mediator able to induce IDO activity, as evaluated by HPLC. TNFα and LPS exerted a synergistic action with IFNγ for IDO activation. In conclusion, MSC express functional key receptors involved in inflammatory response. Their activation modulates properties of MSC into opposite ways: taking part of the inflammatory process through secretion of inflammatory chemokines, or blocking T-cell proliferation through induction of IDO. Such balance should be further studied to favour the development of new clinical trials involving MSC for treatment of aGVHD.

Background & Aim Although native bone marrow-derived mesenchymal stromal cells (BM-MSC) have been described to express CD40, how CD40 is regulated on MSC, and whether CD40 signaling affects MSC activation and function remain unknown. We recently demonstrated that adipose-derived stromal cells (ASC) exhibit a stronger capacity to inhibit immune response than BM-MSC, supporting their use for clinical applications to treat inflammatory diseases and degenerative disorders. Moreover, obesity is associated with adipose tissue inflammation and recruitment of immune cells that could in turn affect the phenotype of native ASC in situ. We thus decided to explore CD40 regulation, signalization, and function on native and in vitro-expanded ASC. Methods, Results & Conclusion Adipose Tissue Stromal Vascular Fractions (ATSVF) were obtained from obese and non-obese donors during lipectomy. Comparison of gene expression was carried out by using RNA sequencing. Some gene expression levels were assessed by QPCR on the total cells or on the CD146− CD34+ Lin− purified ASC. Next, ASC isolated by adhesion in culture were submitted to various stimuli or co-culture with activated CD4+ T cells. Changes in gene expression and secretion profiles were studied by Q-PCR and ELISA. Neutrophil migration was measured through Transwell and involvement of the candidate pathways was assessed by using specific siRNAs. We first revealed through gene expression profiling that the inflammatory signature found in adipose tissue stromal vascular fractions from obese patients included an upregulation of CD40L and was associated with a simultaneous induction of CD40 expression on native ASC. We further confirmed that inflammatory cytokines and direct contact with activated CD4+ T cells upregulated membrane CD40 expression on in vitro-expanded ASC. Stimulation of ASC by CD40L induced a modification of their gene expression profile evaluated by RNAseq reflecting an activation of NF-kB pathway. In agreement, we confirmed by DNA-binding ELISA, the activation of NF-kB pathways by CD40L stimulation. More specifically we focused on the strong induction of inflammatory chemokines, in particular IL-8, triggered on ASC by CD4+ T cells in a CD40/CD40L-dependant manner. Accordingly, CD40L-stimulated ASC recruited more efficiently neutrophils. Overall, these data strongly suggest an interaction between CD4+ T cells, ASC, and neutrophils within inflammatory sites. This observation must be kept in mind when using ASC to treat inflammatory diseases.