Plitidepsin as an Immunomodulator against Respiratory Viral Infections.

Plitidepsin is a host-targeted compound known for inducing a strong anti-SARS-CoV-2 activity, as well as for having the capacity of reducing lung inflammation. Because IL-6 is one of the main cytokines involved in acute respiratory distress syndrome, the effect of plitidepsin in IL-6 secretion in different in vitro and in vivo experimental models was studied. A strong plitidepsin-mediated reduction of IL-6 was found in human monocyte-derived macrophages exposed to nonproductive SARS-CoV-2. In resiquimod (a ligand of TLR7/8)-stimulated THP1 human monocytes, plitidepsin-mediated reductions of IL-6 mRNA and IL-6 levels were also noticed. Additionally, although resiquimod-induced binding to DNA of NF-κB family members was unaffected by plitidepsin, a decrease in the regulated transcription by NF-κB (a key transcription factor involved in the inflammatory cascade) was observed. Furthermore, the phosphorylation of p65 that is required for full transcriptional NF-κB activity was significantly reduced by plitidepsin. Moreover, decreases of IL-6 levels and other proinflammatory cytokines were also seen in either SARS-CoV-2 or H1N1 influenza virus-infected mice, which were treated at low enough plitidepsin doses to not induce antiviral effects. In summary, plitidepsin is a promising therapeutic agent for the treatment of viral infections, not only because of its host-targeted antiviral effect, but also for its immunomodulatory effect, both of which were evidenced in vitro and in vivo by the decrease of proinflammatory cytokines.

Unraveling the antiviral activity of plitidepsin against SARS-CoV-2 by subcellular and morphological analysis.

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation elongation factor 1 alpha) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

Pretransplant adaptive NKG2C+ NK cells protect against cytomegalovirus infection in kidney transplant recipients.

Cytomegalovirus (CMV) infection constitutes a complication for kidney transplant recipients (KTR) and CMV-specific T cells reduce the risk of viral replication in seropositive patients. CMV promotes the adaptive differentiation and expansion of an NK cell subset, hallmarked by expression of the CD94/NKG2C receptor with additional characteristic features. We previously reported an association of pretransplant NKG2C+ NK cells with a reduced incidence of CMV infection. We have strengthened the analysis in cryopreserved peripheral blood mononuclear cells from an enlarged KTR cohort (n = 145) with homogeneous immunosuppression, excluding cases at low risk of infection (ie, CMV D-R-) or receiving antiviral prophylaxis. Moreover, adaptive NKG2C+ NK cell-associated markers (ie, NKG2A, CD57, Immunoglobulin-like transcript 2 [LIR1 or LILRB1], FcεRI γ chain, and Prolymphocytic Leukemia Zinc Finger transcription factor) as well as T lymphocyte subsets were assessed by multicolor flow cytometry. The relation of NKG2C+ NK cells with T cells specific for CMV antigens was analyzed in pretransplant patients (n = 29) and healthy controls (n = 28). Multivariate Cox regression and Kaplan-Meier analyses supported that NKG2C+ NK cells bearing adaptive markers were specifically associated with a reduced incidence of posttransplant symptomatic CMV infection; no correlation between NKG2C+ NK cells and CMV-specific T cells was observed. These results support that adaptive NKG2C+ NK cells contribute to control CMV infection in KTR.

Regulatory dendritic cells for human organ transplantation.

Current immunosuppressive (IS) regimens used to prevent organ allograft rejection have well-recognized side effects, that include enhanced risk of infection and certain types of cancer, metabolic disorders, cardiovascular disease, renal complications and failure to control chronic allograft rejection. The life-long dependency of patients on these IS agents reflects their inability to induce donor-specific tolerance. Extensive studies in rodent and non-human primate models have demonstrated the ability of adoptively-transferred regulatory immune cells (either regulatory myeloid cells or regulatory T cells) to promote transplant tolerance. Consequently, there is considerable interest in the potential of regulatory immune cell therapy to allow safe minimization/complete withdrawal of immunosuppression and the promotion of organ transplant tolerance in the clinic. Here, we review the properties of regulatory dendritic cells (DCreg) with a focus on the approaches being taken to generate human DCreg for clinical testing. We also document the early phase clinical trials that are underway to assess DCreg therapy in clinical organ transplantation as well as in autoimmune disorders.

Peripheral blood lymphocyte subsets change after steroid withdrawal in renal allograft recipients: a prospective study.

Several studies have assessed clinical outcomes after steroid withdrawal (SW) in kidney transplant (KT) recipients, but little is known about its potential impact on lymphocyte subpopulations. We designed a prospective study to evaluate the long-term impact of SW in 19 KT recipients compared to 16 KT recipients without changes in immunosuppression (steroid maintenance, SM). We assessed renal function, presence of HLA antibodies and peripheral blood lymphocyte subsets at time of inclusion, and 3, 12 and 24 months later. The immunophenotype of 20 healthy subjects was also analyzed. Serum creatinine and proteinuria remained stable in SW and SM patients. SW did not associate with generation of de novo donor-specific antibodies. SW patients showed decreases in T-lymphocytes (p < 0.001), and in the CD4+ T cell subpopulation (p = 0.046). The proportion of B-lymphocytes (p = 0.017), and both naïve and transitional B cells increased compared to SM patients (p < 0.001). Changes in B cell subsets were detected 3 months after SW and persisted for 24 months. No changes were observed in NK cells related to steroid withdrawal. SW patients displayed significant changes in peripheral T and B cell subsets, transitioning to the phenotype detected in healthy subjects. This may be considered as a maintained positive effect of SW previously unnoticed.

Intratumoral delivery of mTORC2-deficient dendritic cells inhibits B16 melanoma growth by promoting CD8(+) effector T cell responses.

Dendritic cells (DC) play a pivotal role in the induction and regulation of immune responses. In cancer, DC-based vaccines have proven to be safe and to elicit protective and therapeutic immunological responses. Recently, we showed that specific mTORC2 (mechanistic target of rapamycin complex 2) deficiency in DC enhances their ability to promote Th1 and Th17 responses after LPS stimulation. In the present study, bone marrow-derived mTORC2-deficient (Rictor(-/-)) DC were evaluated as a therapeutic modality in the murine B16 melanoma model. Consistent with their pro-inflammatory profile (enhanced IL-12p70 production and low PD-L1 expression versus control DC), intratumoral (i.t.) injection of LPS-activated Rictor(-/-) DC slowed B16 melanoma growth markedly in WT C57BL/6 recipient mice. This antitumor effect was abrogated when Rictor(-/-) DC were injected i.t. into B16-bearing Rag(-/-) mice, and also after selective CD8(+) T cell depletion in wild-type hosts in vivo, indicating that CD8(+) T cells were the principal regulators of tumor growth after Rictor(-/-) DC injection. I.t. administration of Rictor(-/-) DC also reduced the frequency of myeloid-derived suppressor cells within tumors, and enhanced numbers of IFNγ(+) and granzyme-B(+) cytotoxic CD8(+) T cells both in the spleens and tumors of treated animals. These data suggest that selective inhibition of mTORC2 activity in activated DC augments their pro-inflammatory and T cell stimulatory profile, in association with their enhanced capacity to promote protective CD8(+) T cell responses in vivo, leading to slowed B16 melanoma progression. These novel findings may contribute to the design of more effective DC-based vaccines for cancer immunotherapy.

mTORC2 Deficiency in Myeloid Dendritic Cells Enhances Their Allogeneic Th1 and Th17 Stimulatory Ability after TLR4 Ligation In Vitro and In Vivo.

The mammalian/mechanistic target of rapamycin (mTOR) is a key integrative kinase that functions in two independent complexes, mTOR complex (mTORC) 1 and mTORC2. In contrast to the well-defined role of mTORC1 in dendritic cells (DC), little is known about the function of mTORC2. In this study, to our knowledge, we demonstrate for the first time an enhanced ability of mTORC2-deficient myeloid DC to stimulate and polarize allogeneic T cells. We show that activated bone marrow-derived DC from conditional Rictor(-/-) mice exhibit lower coinhibitory B7-H1 molecule expression independently of the stimulus and enhanced IL-6, TNF-α, IL-12p70, and IL-23 production following TLR4 ligation. Accordingly, TLR4-activated Rictor(-/-) DC display augmented allogeneic T cell stimulatory ability, expanding IFN-γ(+) and IL-17(+), but not IL-10(+) or CD4(+)Foxp3(+) regulatory T cells in vitro. A similar DC profile was obtained by stimulating Dectin-1 (C-type lectin family member) on Rictor(-/-) DC. Using novel CD11c-specific Rictor(-/-) mice, we confirm the alloreactive Th1 and Th17 cell-polarizing ability of endogenous mTORC2-deficient DC after TLR4 ligation in vivo. Furthermore, we demonstrate that proinflammatory cytokines produced by Rictor(-/-) DC after LPS stimulation are key in promoting Th1/Th17 responses. These data establish that mTORC2 activity restrains conventional DC proinflammatory capacity and their ability to polarize T cells following TLR and non-TLR stimulation. Our findings provide new insight into the role of mTORC2 in regulating DC function and may have implications for emerging therapeutic strategies that target mTOR in cancer, infectious diseases, and transplantation.

Regulatory myeloid cells in transplantation.

Regulatory myeloid cells (RMC) are emerging as novel targets for immunosuppressive (IS) agents and hold considerable promise as cellular therapeutic agents. Herein, we discuss the ability of regulatory macrophages, regulatory dendritic cells, and myeloid-derived suppressor cells to regulate alloimmunity, their potential as cellular therapeutic agents, and the IS agents that target their function. We consider protocols for the generation of RMC and the selection of donor- or recipient-derived cells for adoptive cell therapy. Additionally, the issues of cell trafficking and antigen (Ag) specificity after RMC transfer are discussed. Improved understanding of the immunobiology of these cells has increased the possibility of moving RMC into the clinic to reduce the burden of current IS agents and to promote Ag-specific tolerance. In the second half of this review, we discuss the influence of established and experimental IS agents on myeloid cell populations. IS agents believed historically to act primarily on T cell activation and proliferation are emerging as important regulators of RMC function. Better insights into the influence of IS agents on RMC will enhance our ability to develop cell therapy protocols to promote the function of these cells. Moreover, novel IS agents may be designed to target RMC in situ to promote Ag-specific immune regulation in transplantation and to usher in a new era of immune modulation exploiting cells of myeloid origin.

Regulatory dendritic cell therapy: from rodents to clinical application.

Dendritic cells (DC) are highly-specialized, bone marrow-derived antigen-presenting cells that induce or regulate innate and adaptive immunity. Regulatory or "tolerogenic" DC play a crucial role in maintaining self tolerance in the healthy steady-state. These regulatory innate immune cells subvert naïve or memory T cell responses by various mechanisms. Regulatory DC (DCreg) also exhibit the ability to induce or restore T cell tolerance in many animal models of autoimmune disease or transplant rejection. There is also evidence that adoptive transfer of DCreg can regulate T cell responses in non-human primates and humans. Important insights gained from in vitro studies and animal models have led recently to the development of clinical grade human DCreg, with potential to treat autoimmune disease or enhance transplant survival while reducing patient dependency on immunosuppressive drugs. Phase I trials have been conducted in type-1 diabetes and rheumatoid arthritis, with results that emphasize the feasibility and safety of DCreg therapy. This mini-review will outline how observations made using animal models have been translated into human use, and discuss the challenges faced in further developing this form of regulatory immune cell therapy in the fields of autoimmunity and transplantation.

Differential effects of monophosphoryl lipid A and cytokine cocktail as maturation stimuli of immunogenic and tolerogenic dendritic cells for immunotherapy.

Immunotherapy using monocyte-derived dendritic cells (MDDC) is increasingly being considered as alternative therapeutic approach in cancer, infectious diseases and also in autoimmunity when patients are not responsive to conventional treatments. In general, generation of MDDC from monocytes is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the culture to obtain mature DCs suitable for therapy. For DC maturation, different combinations of pro-inflammatory mediators and Toll-like receptor ligands have been tested, obtaining DCs that differ in their properties and the type of immune response they promote. Therefore, it is necessary to find an optimal cytokine environment for DC maturation to obtain a cellular product suitable for DC-based immunotherapeutic protocols. In this study, we have evaluated in vitro the effects of different maturation stimuli on the viability, phenotype, cytokine profile, stability and functionality of immunogenic and tolerogenic (1α,25-dihydroxyvitamin D(3)-treated) MDDC. Maturation was induced using the clinical grade TLR4-agonist: monophosphoryl lipid A (LA), compared to the traditional cytokine cocktail (CC; clinical grade TNF-α, IL-1ß, PGE2) and a combination of both. Our results showed the combination of CC+LA rendered a potent immunogenic DC population that induced the production of IFN-γ and IL-17 in allogeneic co-cultures, suggesting a Th17 polarization. Moreover, these immunogenic DCs showed a high surface expression of CD83, CD86, HLA-DR and secretion of IL-12p70. When aiming to induce tolerance, using LA to generate mature TolDC did not represent a clear advantage, and the stability and the suppressive capability exhibited by CC-matured TolDC may represent the best option. Altogether, these findings demonstrate the relevance of an appropriate maturation stimulus to rationally modulate the therapeutic potential of DCs in immunotherapy.