Ribonuclease activity undermines immune sensing of naked extracellular RNA.

The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

Reverse Phase HPLC Methodology for the Determination of Bay K8644.

Following ICH guidelines for analytical validation, we report a common C18 column stability indicating isocratic reverse phase high performance liquid chromatography method for the determination of the ion channel modulator Bay K8644. Two main forced degradation products and a minor impurity were also tentatively identified by Mass Spectrometry. The mobile phase consisted of a 50/50 acetonitrile/buffer mixture at a flow rate of 2 mL/min. Mean retention time for Bay K8644 was 3.030 minutes. Excellent linearity (r = 0.9998) was achieved in the range 0.10-1.40 µg/mL at 274 nm wavelength. Analytical limits were 16.56 ± 1.04 ng/mL for detection and 55.21 ± 3.48 ng/mL for quantitation respectively. Accuracy and precision studies showed good results (95-105%). Robustness was assessed by varying ±3%, both temperature and flow rate. Five different stress conditions were applied to assess Bay K8644's stability. Only basic and photolytic treatments yielded degradation products, both correctly resolved in a total runtime of 4 minutes. In conclusion, we developed a fast, simple, sensitive, accurate, precise, reliable and stability indicating method for detecting/quantifying Bay K8644, and tentatively characterized its main impurities/forced degradation products.

The intracellular cation channel TMEM176B as a dual immunoregulator.

Characterizing immune regulatory pathways is critical to understand physiological and pathophysiological processes as well as to identify novel immunotherapeutic targets. The cation channel TMEM176B has emerged in the last years as a potential new immunoregulatory player and pharmacological target. Here, we review how expression data, clinical associations of genetic variants and functional studies support a dual role for TMEM176B in regulating immune responses. Thus, TMEM176B can inhibit effector immune responses in some settings whereas it may also promote immunity by supporting antigen presentation in others. We also discuss a potential role for TMEM176B in regulating type 2 and 3 immunity and comment recent data on modulation of DC biology and inflammasome activation as well as CD8+ T cell responses. Understanding the role of TMEM176B in immunity is critical to propose rational pharmacological approaches targeting this channel.

PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical ß-coronavirus disease.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2-related murine ß-coronavirus. Tmem176b-/- mice infected with murine ß-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical ß-coronavirus disease.

Federation of Clinical Immunology Societies Goes South 2021: advanced course on molecular and cellular translational immunology.

The Federation of Clinical Immunology Societies (FOCIS) regularly organizes scientific meetings to foster advances in immunology. A new event of this type is FOCIS Goes South, a course and workshop organized by FOCIS Centers of Excellence (FCEs) from across Latin America, which consists of a course on advanced immunology, a flow cytometry workshop and seminars on cutting-edge research in autoimmunity, tolerance, cancer, infectious diseases and vaccines. Due to the COVID-19 pandemic, the second version of FOCIS Goes South, hosted by the Millennium Institute on Immunology and Immunotherapy in Chile, took place virtually from 15 to 18 November 2021, with more than 950 registered participants. The present article summarizes the key findings and insights discussed at FOCIS Goes South 2021.

The Paradoxical Roles of Inflammation during PD-1 Blockade in Cancer.

Recent studies have reported paradoxical roles of inflammation in tumor immunity triggered by PD-1 checkpoint antibody (Ab) blockade. Here, we elaborate on this controversy and propose a new perspective that might help understand this paradox. Since inflammatory cytokines and PD-1 blockade are known to target different subsets of exhausted CD8+ T cells, we propose that the timing at which anti-PD-1 Ab therapy and cytokine modulation occur might determine the fate of exhausted CD8+ T cells and perhaps, the clinical outcome of immunotherapeutic modalities.

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome.

A major proportion of extracellular RNAs (exRNAs) do not copurify with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes are probably not silent from an immunological perspective and could possibly be decoded as damage-associated molecular patterns.

Role of indoleamine 2,3-dioxygenase in testicular immune-privilege.

Male meiotic germ cell including the spermatozoa represent a great challenge to the immune system, as they appear long after the establishment of normal immune tolerance mechanisms. The capacity of the testes to tolerate autoantigenic germ cells as well as survival of allogeneic organ engrafted in the testicular interstitium have led to consider the testis an immunologically privileged site. Disruption of this immune privilege following trauma, tumor, or autoimmune orchitis often results in male infertility. Strong evidence indicates that indoleamine 2,3-dioxygenase (IDO) has been implicated in fetal and allograft tolerance, tumor immune resistance, and regulation of autoimmune diseases. IDO and tryptophan 2,3-dioxygenase (TDO) catalyze the same rate-limiting step of tryptophan metabolism along a common pathway, which leads to tryptophan starvation and generation of catabolites collectively known as kynurenines. However, the relevance of tryptophan metabolism in testis pathophysiology has not yet been explored. Here we assessed the in vivo role of IDO/TDO in experimental autoimmune orchitis (EAO), a model of autoimmune testicular inflammation and immunologically impaired spermatogenesis. EAO was induced in adult Wistar rats with testicular homogenate and adjuvants. Control (C) rats injected with saline and adjuvants and normal untreated rats (N) were also studied. mRNA expression of IDO decreased in whole testes and in isolated Sertoli cells during EAO. TDO and IDO localization and level of expression in the testis were analyzed by immunostaining and Western blot. TDO is expressed in granulomas from EAO rats, and similar protein levels were observed in N, C, and EAO groups. IDO was detected in mononuclear and endothelial cells and reduced IDO expression was detected in EAO group compared to N and C rats. This phenomenon was concomitant with a significant reduction of IDO activity in EAO testis measured by tryptophan and kynurenine concentrations (HPLC). Finally, in vivo inhibition of IDO with 1-methyl-tryptophan increased severity of the disease, demonstrating down regulation of IDO-based tolerance when testicular immune regulation was disrupted. We present evidence that an IDO-based mechanism is involved in testicular immune privilege.

Targeting TMEM176B Enhances Antitumor Immunity and Augments the Efficacy of Immune Checkpoint Blockers by Unleashing Inflammasome Activation.

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8+ T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1ß activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8+ T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers.

Pro-inflammatory Ca++-activated K+ channels are inhibited by hydroxychloroquine.

Antimalarials have demonstrated beneficial effects in Systemic Lupus Erithematosus and Rheumatoid Arthritis. However, the mechanisms and the molecular players targeted by these drugs remain obscure. Although hydroxychloroquine (HCQ) is a known ion channel inhibitor, this property has not been linked to its anti-inflammatory effects. We aimed to study whether HCQ inhibits pro-inflammatory ion channels. Electrophysiology experiments demonstrated that HCQ inhibited Ca++-activated K+ conductance in THP-1 macrophages in a dose-dependent manner. In macrophages, ATP-induced K+ efflux plays a key role in activating the NLRP3 inflammasome. ATP-induced IL-1beta secretion was controlled by the KCa1.1 inhibitor iberiotoxin. NS1619 and NS309 (KCa1.1 and KCa3.1 activators respectively) induced the secretion of IL-1beta. This effect was inhibited by HCQ and also by iberiotoxin and clotrimazol (KCa3.1 inhibitor), arguing against off-target effect. In vitro, HCQ inhibited IL-1beta and caspase 1 activation induced by ATP in a dose-dependent manner. HCQ impaired K+ efflux induced by ATP. In vivo, HCQ inhibited caspase 1-dependent ATP-induced neutrophil recruitment. Our results show that HCQ inhibits Ca++-activated K+ channels. This effect may lead to impaired inflammasome activation. These results are the basis for i) a novel anti-inflammatory mechanism for HCQ and ii) a new strategy to target pro-rheumatic Ca++-activated K+ channels.

Comparative Study of the Immunoregulatory Capacity of In Vitro Generated Tolerogenic Dendritic Cells, Suppressor Macrophages, and Myeloid-Derived Suppressor Cells.

BACKGROUND: Regulatory myeloid cell (RMC) therapy is a promising strategy for the treatment of immunological disorders such as autoimmune disease and allograft transplant rejection. Various RMC subsets can be derived from total bone marrow using different protocols, but their phenotypes often overlap, raising questions about whether they are truly distinct. METHODS: In this study, we directly compared the phenotype and function of 3 types of RMCs, tolerogenic dendritic cells, suppressor macrophages, and myeloid-derived suppressor cells, generated in vitro from the same mouse strain in a single laboratory. RESULTS: We show that the 3 RMC subsets tested in this study share some phenotypic markers, suppress T cell proliferation in vitro and were all able to prolong allograft survival in a model of skin transplantation. However, our results highlight distinct mechanisms of action that are specific to each cell population. CONCLUSIONS: This study shows for the first time a side-by-side comparison of 3 types of RMCs using the same phenotypic and functional assays, thus providing a robust analysis of their similarities and differences.

Generation and Characterization of Mouse Regulatory Macrophages.

In the last years, cell therapy has become a promising approach to therapeutically manipulate immune responses in autoimmunity, cancer, and transplantation. Several types of lymphoid and myeloid cells origin have been generated in vitro and tested in animal models. Their efficacy to decrease pharmacological treatment has successfully been established. Macrophages play an important role in physiological and pathological processes. They represent an interesting cell population due to their high plasticity in vivo and in vitro. Here, we describe a protocol to differentiate murine regulatory macrophages in vitro from bone marrow precursors. We also describe several methods to assess macrophage classical functions, as their bacterial killing capacity and antigen endocytosis and degradation. Importantly, regulatory macrophages also display suppressive characteristics, which are addressed by the study of their hypostimulatory T lymphocyte capacity and polyclonal T lymphocyte activation suppression.

Combining autologous dendritic cell therapy with CD3 antibodies promotes regulatory T cells and permanent islet allograft acceptance.

Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation.

Evaluation of the therapeutic potential of bone marrow-derived myeloid suppressor cell (MDSC) adoptive transfer in mouse models of autoimmunity and allograft rejection.

Therapeutic use of immunoregulatory cells represents a promising approach for the treatment of uncontrolled immunity. During the last decade, myeloid-derived suppressor cells (MDSC) have emerged as novel key regulatory players in the context of tumor growth, inflammation, transplantation or autoimmunity. Recently, MDSC have been successfully generated in vitro from naive mouse bone marrow cells or healthy human PBMCs using minimal cytokine combinations. In this study, we aimed to evaluate the potential of adoptive transfer of such cells to control auto- and allo-immunity in the mouse. Culture of bone marrow cells with GM-CSF and IL-6 consistently yielded a majority of CD11b+Gr1hi/lo cells exhibiting strong inhibition of CD8+ T cell proliferation in vitro. However, adoptive transfer of these cells failed to alter antigen-specific CD8+ T cell proliferation and cytotoxicity in vivo. Furthermore, MDSC could not prevent the development of autoimmunity in a stringent model of type 1 diabetes. Rather, loading the cells prior to injection with a pancreatic neo-antigen peptide accelerated the development of the disease. Contrastingly, in a model of skin transplantation, repeated injection of MDSC or single injection of LPS-activated MDSC resulted in a significant prolongation of allograft survival. The beneficial effect of MDSC infusions on skin graft survival was paradoxically not explained by a decrease of donor-specific T cell response but associated with a systemic over-activation of T cells and antigen presenting cells, prominently in the spleen. Taken together, our results indicate that in vitro generated MDSC bear therapeutic potential but will require additional in vitro factors or adjunct immunosuppressive treatments to achieve safe and more robust immunomodulation upon adoptive transfer.

Carbon monoxide decreases endosome-lysosome fusion and inhibits soluble antigen presentation by dendritic cells to T cells.

Heme oxygenase-1 (HO-1) inhibits immune responses and inflammatory reactions via the catabolism of heme into carbon monoxide (CO), Fe(2+) , and biliverdin. We have previously shown that either induction of HO-1 or treatment with exogenous CO inhibits LPS-induced maturation of dendritic cells (DCs) and protects in vivo and in vitro antigen-specific inflammation. Here, we evaluated the capacity of HO-1 and CO to regulate antigen presentation on MHC class I and MHC class II molecules by LPS-treated DCs. We observed that HO-1 and CO treatment significantly inhibited the capacity of DCs to present soluble antigens to T cells. Inhibition was restricted to soluble OVA protein, as no inhibition was observed for antigenic OVA-derived peptides, bead-bound OVA protein, or OVA as an endogenous antigen. Inhibition of soluble antigen presentation was not due to reduced antigen uptake by DCs, as endocytosis remained functional after HO-1 induction and CO treatment. On the contrary, CO significantly reduced the efficiency of fusion between late endosomes and lysosomes and not by phagosomes and lysosomes. These data suggest that HO-1 and CO can inhibit the ability of LPS-treated DCs to present exogenous soluble antigens to naïve T cells by blocking antigen trafficking at the level of late endosome-lysosome fusion.

Tolerogenic dendritic cells and negative vaccination in transplantation: from rodents to clinical trials.

The use of immunosuppressive (IS) drugs to treat transplant recipients has markedly reduced the incidence of acute rejection and early graft loss. However, such treatments have numerous adverse side effects and fail to prevent chronic allograft dysfunction. In this context, therapies based on the adoptive transfer of regulatory cells are promising strategies to induce indefinite transplant survival. The use of tolerogenic dendritic cells (DC) has shown great potential, as preliminary experiments in rodents have demonstrated that administration of tolerogenic DC prolongs graft survival. Recipient DC, Donor DC, or Donor Ag-pulsed recipient DC have been used in preclinical studies and administration of these cells with suboptimal immunosuppression increases their tolerogenic potential. We have demonstrated that autologous unpulsed tolerogenic DC injected in the presence of suboptimal immunosuppression are able to induce Ag-specific allograft tolerance. We derived similar tolerogenic DC in different animal models (mice and non-human primates) and confirmed their protective abilities in vitro and in vivo. The mechanisms involved in the tolerance induced by autologous tolerogenic DC were also investigated. With the aim of using autologous DC in kidney transplant patients, we have developed and characterized tolerogenic monocyte-derived DC in humans. In this review, we will discuss the preclinical studies and describe our recent results from the generation and characterization of tolerogenic monocyte-derived DC in humans for a clinical application. We will also discuss the limits and difficulties in translating preclinical experiments to theclinic.

Penicillin binding proteins as danger signals: meningococcal penicillin binding protein 2 activates dendritic cells through Toll-like receptor 4.

Neisseria meningitidis is a human pathogen responsible for life-threatening inflammatory diseases. Meningococcal penicillin-binding proteins (PBPs) and particularly PBP2 are involved in bacterial resistance to ß-lactams. Here we describe a novel function for PBP2 that activates human and mouse dendritic cells (DC) in a time and dose-dependent manner. PBP2 induces MHC II (LOGEC50 = 4.7 µg/ml ± 0.1), CD80 (LOGEC50 = 4.88 µg/ml ± 0.15) and CD86 (LOGEC50 = 5.36 µg/ml ± 0.1). This effect was abolished when DCs were co-treated with anti-PBP2 antibodies. PBP2-treated DCs displayed enhanced immunogenic properties in vitro and in vivo. Furthermore, proteins co-purified with PBP2 showed no effect on DC maturation. We show through different in vivo and in vitro approaches that this effect is not due to endotoxin contamination. At the mechanistic level, PBP2 induces nuclear localization of p65 NF-kB of 70.7 ± 5.1% cells versus 12 ± 2.6% in untreated DCs and needs TLR4 expression to mature DCs. Immunoprecipitation and blocking experiments showed thatPBP2 binds TLR4. In conclusion, we describe a novel function of meningococcal PBP2 as a pathogen associated molecular pattern (PAMP) at the host-pathogen interface that could be recognized by the immune system as a danger signal, promoting the development of immune responses.

What is the role of antigen-processing mechanisms in autologous tolerogenic dendritic cell therapy in organ transplantation?

Injection of autologous tolerogenic dendritic cells is a promising strategy to diminish the burden of harmful immunosuppression in clinical transplantation. We discuss the immunoregulatory mechanisms triggered by this approach. Tolerogenic dendritic cells have long been associated with decreased antigen-processing capacities. However, different lines of evidence led us to propose that injected autologous dendritic cells may need to process donor antigens from graft passenger leukocytes. It is known that drugs such as calcineurin inhibitors can interfere with antigen processing. Indeed, this issue is of the most importance to rationalize the translation of autologous tolerogenic dendritic cell therapy to the clinic.

Preparation of mouse bone marrow-derived dendritic cells with immunoregulatory properties.

Tolerogenic dendritic cells (Tol-DCs) are critical players in physiological tolerance. Moreover, they also play a role in immune regulation both in a pathophysiological context and when used as therapeutic tools in cell therapy strategies. Here, we describe a protocol to differentiate murine Tol-DCs from bone marrow precursors in vitro. Importantly, Tol-DCs actively suppress T cells stimulated with immunogenic allogeneic DCs. Indeed, Tol-DCs generated using this approach can be useful in studying and characterising the immunoregulatory pathways of Tol-DC and in developing Tol-DC-based cell therapy protocols using in vivo models.

Negative vaccination by tolerogenic dendritic cells in organ transplantation.

PURPOSE OF REVIEW: We discussed the use of autologous tolerogenic dendritic cell (Tol-DC) therapy in organ transplantation, with a particular emphasis on illustrating the reasons why it is a clinically relevant approach and interpreting the experimental data that support this strategy. RECENT FINDINGS: Various parameters are critical for engineering Tol-DCs as a therapeutic tool to manipulate antigen-specific immune responses. Our group has shown that in rats, mice and nonhuman primates, bone marrow progenitors cultured with low doses of granulocyte macrophage colony-stimulating factor can generate Tol-DCs. Injection of autologous Tol-DCs (the same strain as the recipient) is able to significantly prolong allograft survival. Autologous Tol-DCs are more effective than allogeneic Tol-DCs in prolonging allograft survival. Although the reason of this difference remains unclear, it indicates the practical advantages of autologous Tol-DCs as a therapeutic tool in a clinical setting. When autologous Tol-DCs (not pulsed with donor antigens) are administered along with suboptimal immunosuppression treatment, a synergistic effect is achieved, resulting in donor-specific allograft tolerance. SUMMARY: Autologous Tol-DC therapy is a promising approach to improve long-term allograft survival. This strategy may also help reduce the immunosuppressive load in grafted patients and, therefore, limit the harmful effects of immunosuppressive agents.