The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells.

The TH9 subset of helper T cells was initially shown to contribute to the induction of autoimmune and allergic diseases, but subsequent evidence has suggested that these cells also exert antitumor activities. However, the molecular events that account for their effector properties are elusive. Here we found that the transcription factor IRF1 enhanced the effector function of TH9 cells and dictated their anticancer properties. Under TH9-skewing conditions, interleukin 1ß (IL-1ß) induced phosphorylation of the transcription factor STAT1 and subsequent expression of IRF1, which bound to the promoters of Il9 and Il21 and enhanced secretion of the cytokines IL-9 and IL-21 from TH9 cells. Furthermore, IL-1ß-induced TH9 cells exerted potent anticancer functions in an IRF1- and IL-21-dependent manner. Our findings thus identify IRF1 as a target for controlling the function of TH9 cells.

Pathogen exposure influences immune parameters around weaning in pigs reared in commercial farms.

BACKGROUND: Multiple antigenic stimulations are crucial to immune system training during early post-natal life. These stimulations can be either due to commensals, which accounts for the acquisition and maintenance of tolerance, or to pathogens, which triggers immunity. In pig, only few works previously explored the influence of natural exposition to pathogens upon immune competence. We propose herein the results of a multicentric, field study, conducted on 265 piglets exposed to contrasted pathogen levels in their living environment. Piglets were housed in 15 different commercial farms, sorted in two groups, low (HSLOW)- and high (HSHIGH)-health status farms, depending on their recurrent exposition to five common swine pathogens. RESULTS: Using animal-based measures, we compared the immune competence and growth performances of HSLOW and HSHIGH pigs around weaning. As expected, we observed a rise in the number of circulating leucocytes with age, which affected different cell populations. Monocyte, antigen-experienced and cytotoxic lymphocyte subpopulation counts were higher in piglets reared in HSLOW farms as compared to their HSHIGH homologs. Also, the age-dependent evolution in γδ T cell and neutrophil counts was significantly affected by the health status. With age, circulating IFNα level decreased and IgM level increased while being greater in HSLOW piglets at any time. After weaning, LPS-stimulated blood cells derived from HSLOW piglets were more prone to secrete IL-8 than those derived from HSHIGH pigs did. Monocytes and granulocytes issued from HSLOW pigs also exhibited comparable phagocytosis capacity. Altogether our data emphasize the more robust immunophenotype of HSLOW piglets. Finally, piglets raised under higher pathogen pressure grew less than HSHIGH piglets did and exhibited a different metabolic profile. The higher cost of the immune responses associated with the low farm health status may account for lower HSLOW piglet performances. CONCLUSIONS: Altogether, our data, obtained in field conditions, provide evidence that early exposure to pathogens shapes the immune competence of piglets. They also document the negative impact of an overstimulation of the immune system on piglets' growth.

Stat3 and Gfi-1 transcription factors control Th17 cell immunosuppressive activity via the regulation of ectonucleotidase expression.

Although Th17 cells are known to promote tissue inflammation and autoimmunity, their role during cancer progression remains elusive. Here, we showed that in vitro Th17 cells generated with the cytokines IL-6 and TGF-ß expressed CD39 and CD73 ectonucleotidases, leading to adenosine release and the subsequent suppression of CD4(+) and CD8(+) T cell effector functions. The IL-6-mediated activation of the transcription factor Stat3 and the TGF-ß-driven downregulation of Gfi-1 transcription factor were both essential for the expression of ectonucleotidases during Th17 cell differentiation. Stat3 supported whereas Gfi-1 repressed CD39 and CD73 expression by binding to their promoters. Accordingly, Th17 cells differentiated with IL-1ß, IL-6, and IL-23 but without TGF-ß did not express ectonucleotidases and were not immunosuppressive. Finally, adoptive transfer of Th17 cells induced by TGF-ß and IL-6 promoted tumor growth in a CD39-dependent manner. Thus, ectonucleotidase expression supports the immunosuppressive fate of Th17 cells in cancer.

Calreticulin exposure dictates the immunogenicity of cancer cell death.

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy.

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen-specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.

In situ immune response after neoadjuvant chemotherapy for breast cancer predicts survival.

Accumulating preclinical evidence suggests that anticancer immune responses contribute to the success of chemotherapy. However, the predictive value of tumour-infiltrating lymphocytes after neoadjuvant chemotherapy for breast cancer remains unknown. We hypothesized that the nature of the immune infiltrate following neoadjuvant chemotherapy would predict patient survival. In a series of 111 consecutive HER2- and a series of 51 non-HER2-overexpressing breast cancer patients treated by neoadjuvant chemotherapy, we studied by immunohistochemistry tumour infiltration by FOXP3 and CD8 T lymphocytes before and after chemotherapy. Kaplan-Meier analysis and Cox modelling were used to assess relapse-free survival (RFS) and overall survival (OS). A predictive scoring system using American Joint Committee on Cancer (AJCC) pathological staging and immunological markers was created. Association of high CD8 and low FOXP3 cell infiltrates after chemotherapy was significantly associated with improved RFS (p = 0.02) and OS (p = 0.002), and outperformed classical predictive factors in multivariate analysis. A combined score associating CD8/FOXP3 ratio and pathological AJCC staging isolated a subgroup of patients with a long-term overall survival of 100%. Importantly, this score also identified patients with a favourable prognosis in an independent cohort of HER2-negative breast cancer patients. These results suggest that immunological CD8 and FOXP3 cell infiltrate after treatment is an independent predictive factor of survival in breast cancer patients treated with neoadjuvant chemotherapy and provides new insights into the role of the immune milieu and cancer.

Optimization of an O2-balanced bioartificial pancreas for type 1 diabetes using statistical design of experiment.

A bioartificial pancreas (BAP) encapsulating high pancreatic islets concentration is a promising alternative for type 1 diabetes therapy. However, the main limitation of this approach is O2 supply, especially until graft neovascularization. Here, we described a methodology to design an optimal O2-balanced BAP using statistical design of experiment (DoE). A full factorial DoE was first performed to screen two O2-technologies on their ability to preserve pseudo-islet viability and function under hypoxia and normoxia. Then, response surface methodology was used to define the optimal O2-carrier and islet seeding concentrations to maximize the number of viable pseudo-islets in the BAP containing an O2-generator under hypoxia. Monitoring of viability, function and maturation of neonatal pig islets for 15 days in vitro demonstrated the efficiency of the optimal O2-balanced BAP. The findings should allow the design of a more realistic BAP for humans with high islets concentration by maintaining the O2 balance in the device.

CD4+CD25+ Tregs control the TRAIL-dependent cytotoxicity of tumor-infiltrating DCs in rodent models of colon cancer.

Tumors that progress do so via their ability to escape the antitumor immune response through several mechanisms, including developing ways to induce the differentiation and/or recruitment of CD4(+)CD25(+) Tregs. The Tregs, in turn, inhibit the cytotoxic function of T cells and NK cells, but whether they have an effect on the cytotoxic function of tumor-infiltrating DCs (TIDCs) has not been determined. Here we have shown, in 2 rodent models of colon cancer, that CD4(+)CD25(+) Tregs inhibit the ability of CD11b(+) TIDCs to mediate TNF-related apoptosis-inducing ligand-induced (TRAIL-induced) tumor cell death. In both models of cancer, combination treatment with Mycobacterium bovis Bacillus Calmette-Guérin (BCG), which activates the innate immune system via TLR2, TLR4, and TLR9, and cyclophosphamide (CTX), which depletes Tregs, eradicated the tumors. Further analysis revealed that the treatment led to a marked increase in the number of CD11b(+) TIDCs that killed the tumor cells via a TRAIL-dependent mechanism. Furthermore, acquisition of TRAIL expression by the CD11b(+) TIDCs was induced by BCG and dependent on signaling through TLR2, TLR4, and TLR9. In vivo transfer of Tregs abrogated the ability of BCG to induce CD11b(+) TIDCs to express TRAIL and thereby nullified the efficacy of the CTX-BCG treatment. Our data have therefore delineated what we believe to be a novel mechanism by which Tregs inhibit the antitumor immune response.

Presence of Foxp3 expression in tumor cells predicts better survival in HER2-overexpressing breast cancer patients treated with neoadjuvant chemotherapy.

The Forkhead Box Protein 3 is highly expressed not only in regulatory T cells, but also in tumor cells, acting as a transcriptional repressor of breast oncogenes including HER2. We investigated the prognostic significance of Foxp3 expression in cancer cells in a large cohort of patients with HER2-overexpressing breast carcinoma treated with neoadjuvant chemotherapy. Foxp3-positive tumor cells were detected by immunohistochemistry in 103 patients with primary invasive HER2-overexpressing breast carcinoma, and treated with neoadjuvant chemotherapy, with or without trastuzumab. Kaplan-Meier analysis and Cox regression model were used to assess relapse-free and overall survival, respectively, and according to the presence or the absence of Foxp3 expression in tumor cells. Breast cancer cells were Foxp3+ in 57% of tumors. Foxp3 expression in breast cancer cells was associated with better relapse-free (P = 0.005) and overall survival (P = 0.03). By multivariate analysis, the presence of Foxp3+ tumor cells produced an independent prognostic factor for both better relapse-free (P = 0.006) and overall survival (P = 0.03). These findings indicate that the presence of Foxp3+ tumor cells represents a new independent prognostic factor of improved outcome in HER2-overexpressing breast carcinoma, which could help identify high-risk patients for additional therapies after neoadjuvant chemotherapy.

Killer dendritic cells: IKDC and the others.

Tumors can regress as a result of invading myeloid and lymphoid cells that act in concert. Although the myeloid cells are widely recognized as antigen presenters and lymphoid cells as classical effectors, recent evidence revealed the capacity of dendritic cells (DC) to kill tumor cells. The functional concept of 'natural killer (NK) myeloid DC' is supported by mouse and human in vitro data that may be clinically relevant because human killer DC can contribute to tumor shrinking during topical therapy with toll-like receptor (TLR) agonists. Whether tumor killing by DC is a 'catalyzing' step for efficient crosspresentation and/or a promoting step for an immunogenic cell death pathway remains an open question. We also discuss how interferon-producing killer DC (IKDC) may participate in the control of tumor progression.

Dendritic cells and innate defense against tumor cells.

Tumor growth results from a delicate balance between intrinsic dysregulation of oncogenes, tumor suppressor and stability genes counteracted by extrinsic defenses composed of immune cells shaping tumor immunogenicity. Although immune subversion might be the ultimate outcome of this process, a complex network of cellular interactions take place eventually leading to tumor specific cognate immune responses. The links between innate and cognate antitumor immunity eliciting protective T cell responses are instigated by cytokines, chemokines and damage associated molecular patterns. The intricate differentiation pathway whereby dendritic cells could undergo an efficient maturation program in the tumor microenvironment appears crucial. We will discuss the role of innate effectors and cancer therapies in the process of defense against tumor cells.

[Extracellular vesicles are players of the immune continuum]. / Les vésicules extracellulaires - Un maillon essentiel du système immunitaire.

Regulation of immune responses was among the first functions of extracellular vesicles to be identified, more than twenty years ago. What exactly defines the outcome of an immune response remains a challenging issue. Owing to their reduced size, extracellular vesicles easily diffuse in interstitial and lymphatic fluids, where they can interact with the multiple effectors of the immune system. By accelerating and amplifying immune interactions, these ultra-mobile units may contribute to local and systemic coordination for efficient adaption to external and internal changes. Here we introduce the related ground-breaking studies of extracellular vesicle-mediated immune effects and present ongoing considerations on their potential roles in health and the development of immune disorders.

TITLE: Les vésicules extracellulaires - Un maillon essentiel du système immunitaire. ABSTRACT: L'implication des vésicules extracellulaires dans les échanges immunitaires a été parmi les premières fonctions mises en évidence pour ces vésicules, il y a plus de vingt ans. Du fait de leur petite taille, elles diffusent en effet aisément via les fluides interstitiels et la lymphe où elles interagissent avec les multiples effecteurs du système immunitaire. En accélérant et en amplifiant les échanges, il est concevable que ces unités ultra-mobiles favorisent la concertation entre cellules à l'échelle locale et globale, en réponse aux changements que subit l'organisme, que ceux-ci soient internes ou externes. Ici, vous sont présentées les découvertes clés sur les rôles des vésicules extracellulaires dans l'immunité, dont l'impact sur la santé commence tout juste maintenant à être mesuré.

Molecular and Functional Diversity of Distinct Subpopulations of the Stressed Insulin-Secreting Cell's Vesiculome.

Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cell's vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting toward EV subspecies. Functional in vitro assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response.

Immunogenicity of anthracyclines: moving towards more personalized medicine.

The current method of cancer management takes into account tumor-related factors to predict therapeutic outcome. However, recent evidence indicates that the host immune system also contributes to therapeutic outcome. Here, we highlight anthracyclines, which have been used to treat a broad range of cancers since the 1960s, as an example of an anticancer treatment that can boost the host's immune system to improve the efficacy of chemotherapy. It has recently been revealed that the translocation of calreticulin to the plasma membrane in tumor cells and the release of high-mobility-group box 1 (HMGB1) by tumor cells are two key post-transcriptional events required for the immunogenicity of anthracyclines. These discoveries represent a conceptual advance in the understanding of the mechanisms underlying the immunogenicity of anthracyclines. We review the effects of anthracyclines on the host immune system and discuss how this knowledge can be exploited for anticancer therapy.

Therapy-induced tumor immunosurveillance involves IFN-producing killer dendritic cells.

A unique class of IFN-producing killer dendritic cells (IKDC) resembling natural killer cells has been defined that can recognize and lyse tumor cells through a tumor necrosis factor-related apoptosis-inducing ligand-dependent mechanism. IKDC may mediate the host-dependent antitumor activity of Gleevec/STI571 and other therapeutics that can inhibit the c-kit tyrosine kinase. IKDC represent an important new component of the innate immune system responding to cancer.

Interferon-gamma is produced by another player of innate immune responses: the interferon-producing killer dendritic cell (IKDC).

Interferon-gamma is a key cytokine in tumor immunosurveillance. The recently described interferon-producing killer dendritic cell (IKDC), can be distinguished from other innate effectors by its ability to kill a large variety of tumor cells and to produce high amounts of interferon-gamma after encountering tumors in the absence of exogenous cytokines. The cytotoxic activity of IKDC was unraveled during an efficient immunotherapy combining c-kit tyrosine kinase inhibitors and interleukin-2, and is mediated through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and interferon type IIR.

ß2-adrenergic stimulation of dendritic cells favors IL-10 secretion by CD4+ T cells.

Adrenergic receptor agonists and antagonists are extensively used as drugs in medicine for a broad spectrum of indications. We examined the consequences of ß2-adrenergic stimulation of murine dendritic cells (DCs) on CD4+ T cell activation. We demonstrated in vitro that treatment of LPS-matured DCs with the ß2-agonist salbutamol reduced their ability to trigger OT-II T cell proliferation specific for ovalbumin antigen. Salbutamol also induced a decrease in MHC class II molecule expression by DC through Gi protein activation. Co-culture of CD4+ T cells with salbutamol-conditioned mature DC impaired TNFα and IL-6 secretion while preserving IL-10 production by T cells. Using a vaccination protocol in mice, we showed that salbutamol favored IL-10-producing CD4+ T cells. None of these effects was observed when working with ß2-adrenoreceptor deficient mice. Finally, we suggest that ß2-adrenergic stimulation of DC could be an interesting way to shape CD4+ T cell responses for the purposes of immunotherapy.

ß2-adrenoreceptor stimulation dampens the LPS-induced M1 polarization in pig macrophages.

The cross-talk between sympatho-adreno-medullar axis and innate immunity players was mainly studied in rodents. In intensive husbandry, pigs are exposed to multiple stressors inducing repeated releases of catecholamines that bind to adrenoreceptors (AR) on target cells. Among adrenoreceptors, the ß2-AR is largely expressed by immune cells including macrophages. We report herein on the effects of catecholamines, through ß2-AR stimulation, on pig macrophage functions activated by LPS. ß2-AR stimulation of porcine macrophages prevented the LPS-induced increase in TNFα and IL-8 secretion while increasing IL-10 secretion. In contrast, treatment with a ß2-agonist had no effect on anti-microbial functions. Lastly, ß2-AR stimulation of macrophages reduced the expression of genes up-regulated by LPS. Altogether, we demonstrated that ß2-AR stimulation of porcine macrophages prevented polarization towards a pro-inflammatory phenotype. Since porcine macrophages are a suitable model for human macrophages, our results might be relevant to appreciate catecholamine effects on human macrophages.

Acute social stress-induced immunomodulation in pigs high and low responders to ACTH.

Pig husbandry is known as an intensive breeding system, piglets being submitted to multiple stressful events such as early weaning, successive mixing, crowding and shipping. These stressors are thought to impair immune defences and might contribute, at least partly, to the prophylactic use of antibiotics. Robustness was recently defined as the ability of an individual to express a high-production potential in a wide variety of environmental conditions. Increasing robustness thus appears as a valuable option to improve resilience to stressors and could be obtained by selecting piglets upon their adrenocortical activity. In this study, we aimed at depicting the consequences of an acute social stress on the immune capacity of piglets genetically selected upon divergent hypothalamic-pituitary-adrenocortical (HPA) axis activity. For this purpose, we monitored neuroendocrine and immune parameters, in high- (HPAhi) and low- (HPAlo) responders to ACTH, just before and immediately after a one-hour mixing with unfamiliar conspecifics. As expected, stressed piglets displayed higher levels of circulating cortisol and norepinephrine. Blood cell count analysis combined to flow cytometry revealed a stress-induced leukocyte mobilization in the bloodstream with a specific recruitment of CD8α+ lymphocytes. Besides, one-hour mixing decreased LPS-induced IL-8 and TNFα secretions in whole-blood assays (WBA) and reduced mononuclear cell phagocytosis. Altogether, our data demonstrate that acute social stress alters immune competence of piglets from both groups, and bring new insights in favour of good farming practices. While for most parameters high- and low-responders to ACTH behaved similarly, HPAhi piglets displayed higher number of CD4+ CD8α- T cells, as well as increased cytokine production in WBA (LPS-induced TNFα and PIL-induced IL-8), which could confer them increased resistance to pathogens. Finally, a principal component analysis including all parameters highlighted that overall stress effects were less pronounced on piglets with a strong HPA axis. Thus, selection upon adrenocortical axis activity seems to reduce the magnitude of response to stress and appears as a good tool to increase piglet robustness.