Cutting Edge: mTORC1 Inhibition in Metastatic Breast Cancer Patients Negatively Affects Peripheral NK Cell Maturation and Number.

NK cells are cytotoxic lymphocytes displaying strong antimetastatic activity. Mouse models and in vitro studies suggest a prominent role of the mechanistic target of rapamycin (mTOR) kinase in the control of NK cell homeostasis and antitumor functions. However, mTOR inhibitors are used as chemotherapies in several cancer settings. The impact of such treatments on patients' NK cells is unknown. We thus performed immunophenotyping of circulating NK cells from metastatic breast cancer patients treated with the mTOR inhibitor everolimus over a three-month period. Everolimus treatment resulted in inhibition of mTORC1 activity in peripheral NK cells, whereas mTORC2 activity was preserved. NK cell homeostasis was profoundly altered with a contraction of the NK cell pool and an overall decrease in their maturation. Phenotype and function of the remaining NK cell population was less affected. This is, to our knowledge, the first in vivo characterization of the role of mTOR in human NK cells.

Deletion of Inflammasome Components Is Not Sufficient To Prevent Fatal Inflammation in Models of Familial Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition that occurs in patients with genetic defects of cytotoxicity (familial HLH [FHL]) or secondary to other immunological disorders such as juvenile idiopathic arthritis. HLH is characterized by elevated levels of serum IL-18 and other cytokines. Moreover, a novel clinical entity has been recently identified in which constitutive NLRC4 inflammasome activation leads to severe HLH. Altogether, these clinical observations suggest that inflammasome activation is a central event in the development of all HLH forms and that inflammasome blockade could alleviate inflammation in FHL patients. To formally address this question, we invalidated genes encoding for Caspase-1 or the inflammasome adapter ASC in perforin-deficient mice that were subsequently infected with lymphocytic or mouse choriomeningitis virus as models of FHL. These deletions nearly abrogated IL-18 production occurring during HLH in all models. However, they did not reduce serum IFN-γ levels at the peak of the inflammatory reaction nor did they modulate inflammatory parameters at mid and late stages or fatal outcome. These data show that inflammasome blockade is not sufficient to prevent cytokine storm and lethality in mouse models of FHL and suggest that different pathophysiological mechanisms underlie HLH in genetic defects of cytotoxicity and genetic forms of inflammasome activation.

Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens.

The liver is the largest gland in the human body and functions as an innate immune organ. Liver macrophages called Kupffer cells (KC) constitute the largest group of macrophages in the human body. Innate immune responses involving KC represent the first line of defense against pathogens in the liver. Human monocyte-derived macrophages have been used to characterize inflammasome responses that lead to the release of the proinflammatory cytokines IL-1ß and IL-18, but it has not yet been determined whether human KC contain functional inflammasomes. We show, to our knowledge for the first time, that KC express genes and proteins that make up several different inflammasome complexes. Moreover, activation of KC in response to the absent in melanoma 2 (AIM2) inflammasome led to the production of IL-1ß and IL-18, which activated IL-8 transcription and hepatic NK cell activity, respectively. Other inflammasome responses were also activated in response to selected bacteria and viruses. However, hepatitis B virus inhibited the AIM2 inflammasome by reducing the mRNA stability of IFN regulatory factor 7, which regulated AIM2 transcription. These data demonstrate the production of IL-1ß and IL-18 in KC, suggesting that KC contain functional inflammasomes that could be important players in the innate immune response following certain infections of the liver. We think our findings could potentially aid therapeutic approaches against chronic liver diseases that activate the inflammasome.

Alteration of Natural Killer cell phenotype and function in obese individuals.

Obesity is associated with increased cancer rates and higher susceptibility to infections. The adipose tissue of obese individuals is inflammatory and may negatively impact on innate and adaptive immunity in a systemic way. Here, we explored the phenotype and function of peripheral Natural Killer (NK) cells of patients in correlation with their body mass index (BMI). We found that high BMI was associated with an increased activation status of peripheral NK cells, as measured by surface levels of CD69 and levels of granzyme-B. However, these activated NK cells had an impaired capacity to degranulate or to produce cytokines/chemokines when exposed to tumor cell lines deficient in MHC-I expression or coated with antibodies. This suggests that chronic stimulation of NK cells during obesity may lead to their incapacity to respond normally and eliminate target cells, which could contribute to the greater susceptibility of obese individuals to develop cancers or infectious diseases.

Impaired interferon-alpha production by plasmacytoid dendritic cells after cord blood transplantation in children: implication for post-transplantation toll-like receptor ligand-based immunotherapy.

Plasmacytoid dendritic cells (pDCs) initiate both innate and adaptive immune responses, making them attractive targets for post-transplantation immunotherapy, particularly after cord blood transplantation (CBT). Toll-like receptor (TLR) agonists are currently studied for pDC stimulation in various clinical settings. Their efficacy depends on pDC number and functionality, which are unknown after CBT. We performed a longitudinal study of pDC reconstitution in children who underwent bone marrow transplantation (BMT) and single-unit CBT. Both CBT and unrelated BMT patients received antithymocyte globulin as part of their graft-versus-host disease prophylaxis regimen. pDC blood counts were higher in CBT patients than in healthy volunteers from 2 to 9 months after transplantation, whereas they remained lower in BMT patients. We showed that cord blood progenitors gave rise in vitro to a 500-fold increase in functional pDCs over bone marrow counterparts. Upon stimulation with a TLR agonist, pDCs from both CBT and BMT recipients upregulated T cell costimulatory molecules, whereas interferon-alpha (IFN-α) production was impaired for 9 months after CBT. TLR agonist treatment is thus not expected to induce IFN-α production by pDCs after CBT, limiting its immunotherapeutic potential. Fortunately, in vitro production of large amounts of functional pDCs from cord blood progenitors paves the way for the post-transplantation adoptive transfer of pDCs.

Reconstitution of protective immune responses against cytomegalovirus and varicella zoster virus does not require disease development in pediatric recipients of umbilical cord blood transplantation.

CMV and varicella zoster virus (VZV) are significant causes of morbidity and mortality following umbilical cord blood transplantation (UCBT). However, the kinetics of reconstitution and protective potential of antiviral cell-mediated immune responses following UCBT remain poorly characterized. In this study, the reconstitution of CMV- and VZV-specific T cell responses was assessed using IFN-γ ELISPOT in 28 children who underwent UCBT to treat hematological or inherited disorders. Barely detectable in the first 3 mo posttransplantation, CMV- and VZV-specific T cell responses were observed in 30.4% and 40.3% of study subjects after 36 mo of follow-up. Four of five CMV-seropositive subjects developed detectable levels of circulating CMV DNA (DNAemia), and 5 of 17 VZV-seropositive patients experienced herpes zoster during the posttransplant period. Four CMV-seronegative subjects developed IFN-γ responses against CMV, and four subjects developed a VZV-specific IFN-γ response without clinical signs of infection. No CMV- or VZV-related events were observed in study subjects following the development of CMV- or VZV-specific responses > 150 spot-forming units/10(6) PBMCs, consistent with T cell-mediated protection. Finally, famciclovir prophylaxis did not strictly prevent the reconstitution of the VZV-specific T cell repertoire, because the frequency of T cells producing IFN-γ in response to VZV Ags reached levels consistent with protection in two nonzoster subjects. Monitoring of CMV- and VZV-specific cell-mediated immunity could inform immunocompetence and guide the initiation and cessation of antiherpetic prophylaxis in UCBT recipients.

Engineering a versatile and retrievable cell macroencapsulation device for the delivery of therapeutic proteins.

Encapsulated cell therapy holds a great potential to deliver sustained levels of highly potent therapeutic proteins to patients and improve chronic disease management. A versatile encapsulation device that is biocompatible, scalable, and easy to administer, retrieve, or replace has yet to be validated for clinical applications. Here, we report on a cargo-agnostic, macroencapsulation device with optimized features for protein delivery. It is compatible with adherent and suspension cells, and can be administered and retrieved without burdensome surgical procedures. We characterized its biocompatibility and showed that different cell lines producing different therapeutic proteins can be combined in the device. We demonstrated the ability of cytokine-secreting cells encapsulated in our device and implanted in human skin to mobilize and activate antigen-presenting cells, which could potentially serve as an effective adjuvant strategy in cancer immunization therapies. We believe that our device may contribute to cell therapies for cancer, metabolic disorders, and protein-deficient diseases.

Erratum: Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

[This corrects the article DOI: 10.1016/j.omtm.2022.07.017.].

Post-transcriptional down-regulation of Toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (PDCs) from human umbilical cord blood (UCB) produce lower amounts of IFN-α upon TLR stimulation compared with adult counterparts. This difference may play a role in the low graft-versus-host disease rate after UCB transplantation and in the impaired immune response of the neonate to pathogens. Comparing UCB PDC to their adults counterparts, we found that they exhibited a mature surface phenotype and a normal antigen uptake. They upregulated costimulatory molecules upon activation, although with delayed kinetics. Protein, but not ARN, levels of TLR-9, MyD88, IRAK1 and IRF-7, involved in the TLR-9 signaling pathway were reduced. The expression levels of miR-146a and miR-155, known to be involved in the post-transcriptional down-regulation of immune responses, were higher. These data point out a post-transcriptional down-regulation of the TLR-9/IRF-7 signaling pathway in UCB PDC.

Defects in CD54 and CD86 up-regulation by plasmacytoid dendritic cells during pregnancy.

Physiological modulation of the immune system is required for foetal tolerance during pregnancy. However, this immune regulation might lead to impaired self-defence against pathogens. Indeed, pregnant women are more susceptible to newly encountered viruses comparing to non-pregnant women, as exemplified by the prevalence of severe complications in pregnant women infected with the pandemic influenza virus in 2009. Plasmacytoid dendritic cells (pDCs) are specialized dendritic cells that recognise viral antigens and initiate both innate and adaptive immune responses. We therefore sought to determine whether the number and/or the functions of peripheral blood pDCs are regulated during pregnancy. pDC maturation and interferon (IFN)-α production were analysed in response to Toll-like receptor (TLR) stimulation of peripheral blood mononuclear cells from pregnant and non-pregnant women. Our results reveal that pDC frequency is slightly decreased, while the IFN-α production in response to TLR stimulation increases during pregnancy. Interestingly, the up-regulation of the co-stimulatory receptors CD54 (ICAM1) and CD86 is significantly decreased in pDCs from pregnant women as compared to controls, suggesting a possible impact on T-cell responses. In conclusion, we propose that the modulation of CD54 and CD86 expression on peripheral blood pDCs during pregnancy might decrease the initiation of adaptive antiviral immune responses.

Umbilical cord blood T cells respond against the Melan-A/MART-1 tumor antigen and exhibit reduced alloreactivity as compared with adult blood-derived T cells.

Umbilical cord blood (UCB) is increasingly used as a source of hematopoietic progenitor cells to treat a variety of disorders. UCB transplant is associated with comparatively reduced incidence of graft-versus-host disease, robust graft versus leukemia effect, and relatively high incidence of opportunistic infections, three processes in which donor-derived T lymphocytes are known to be predominantly involved. To examine the differential functionality of UCB T cells, CD8(+) T cells specific for the melanoma-associated HLA-A2-restricted Melan-A(26-35) A27L peptide were isolated from HLA-A2(+) and HLA-A2(-) UCB samples and HLA-A2(+) and HLA-A2(-) adult peripheral blood using A2/Melan-A tetramers. In UCB samples, A2/Melan-A(+) CD8(+) T cells were detected at a frequency of 0.04%, were more frequent in HLA-A2(+) UCB, and were polyclonal and mostly naive. Consistent with Ag-driven expansion, the frequency of A2/Melan-A(+) CD8(+) T cells was increased following stimulation with cognate peptide or polyclonal activation, they acquired cell-surface markers reflective of effector/memory differentiation, their TCR repertoire became oligoclonal, and they expressed cytolytic activity and produced IFN-gamma. Although functional properties of A2/Melan-A(+) CD8(+) T cells derived from HLA-A2(+) UCB resembled those of HLA-A2(+) adult peripheral blood, they were more likely to reach terminal differentiation following polyclonal stimulation and produced less IFN-gamma in response to cognate peptide. A2/Melan-A(+) CD8(+) T cells from HLA-A2(-) UCB were poorly cytolytic, produced little IFN-gamma, and were predominantly monofunctional or nonfunctional. These properties of UCB-derived CD8(+) T cells could contribute to the reduced incidence of graft-versus-host disease and heightened incidence of opportunistic infections observed following UCB transplant.

Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen. We observed that, when encapsulated, the novel myoblast cell line can be efficiently frozen, stored, and thawed, which limits the challenge imposed by the manufacture and supply of encapsulated cell-based therapeutic products. Our results suggest that this versatile allogeneic cell line represents the next step toward a broader development and therapeutic use of ECT.

A Quantitative ELISA Protocol for Detection of Specific Human IgG against the SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with at least 3.8 million deaths to date. For that reason, finding an efficient vaccine for this virus quickly became a global priority. The majority of vaccines now marketed are based on the SARS-CoV-2 spike protein that has been described as the keystone for optimal immunization. In order to monitor SARS-CoV-2 spike-specific humoral responses generated by immunization or infection, we have developed a robust and reproducible enzyme-linked immunosorbent assay (ELISA) protocol. This protocol describes a method for quantitative detection of IgG antibodies against the SARS-CoV-2 spike protein using antigen-coated microtiter plates. Results showed that antibodies could be quantified between the range of 1.953 ng/mL to 500 ng/mL with limited inter- and intra-assay variability.

Local Sustained GM-CSF Delivery by Genetically Engineered Encapsulated Cells Enhanced Both Cellular and Humoral SARS-CoV-2 Spike-Specific Immune Response in an Experimental Murine Spike DNA Vaccination Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with recurrences. Therefore, finding a vaccine for this virus became a priority for the scientific community. The SARS-CoV-2 spike protein has been described as the keystone for viral entry into cells and effective immune protection against SARS-CoV-2 is elicited by this protein. Consequently, many commercialized vaccines focus on the spike protein and require the use of an optimal adjuvant during vaccination. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated a powerful enhancement of acquired immunity against many pathogens when delivered in a sustained and local manner. In this context, we developed an encapsulated cell-based technology consisting of a biocompatible, semipermeable capsule for secretion of GM-CSF. In this study, we investigated whether murine GM-CSF (muGM-CSF) represents a suitable adjuvant for SARS-CoV-2 immunization, and which delivery strategy for muGM-CSF could be most beneficial. To test this, different groups of mice were immunized with intra-dermal (i.d.) electroporated spike DNA in the absence or presence of recombinant or secreted muGM-CSF. Results demonstrated that adjuvanting a spike DNA vaccine with secreted muGM-CSF resulted in enhancement of specific cellular and humoral immune responses against SARS-CoV-2. Our data also highlighted the importance of delivery strategies to the induction of cellular and humoral-mediated responses.

An Experimentally Defined Hypoxia Gene Signature in Glioblastoma and Its Modulation by Metformin.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O2) and to physioxia (5% O2). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O2 to 1% O2. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.

One-Year Follow-Up of Natural Killer Cell Activity in Multiple Myeloma Patients Treated With Adjuvant Lenalidomide Therapy.

Multiple myeloma (MM) is a proliferation of tumoral plasma B cells that is still incurable. Natural killer (NK) cells can recognize and kill MM cells in vitro and can limit MM growth in vivo. Previous reports have shown that NK cell function is impaired during MM progression and suggested that treatment with immunomodulatory drugs (IMIDs) such as lenalidomide (LEN) could enhance it. However, the effects of IMIDs on NK cells have been tested mostly in vitro or in preclinical models and supporting evidence of their effect in vivo in patients is lacking. Here, we monitored NK cell activity in blood samples from 10 MM patients starting after frontline induction chemotherapy (CTX) consisting either of association of bortezomib-lenalidomide-dexamethasone (Velcade Revlimid Dexamethasone) or autologous stem-cell transplantation (SCT). We also monitored NK cell activity longitudinally each month during 1 year, after maintenance therapy with LEN. Following frontline chemotherapy, peripheral NK cells displayed a very immature phenotype and retained poor reactivity toward target cells ex vivo. Upon maintenance treatment with LEN, we observed a progressive normalization of NK cell maturation, likely caused by discontinuation of chemotherapy. However, LEN treatment neither activated NK cells nor improved their capacity to degranulate or to secrete IFN-γ or MIP1-ß following stimulation with MHC-I-deficient or antibody-coated target cells. Upon LEN discontinuation, there was no reduction of NK cell effector function either. These results caution against the use of LEN as single therapy to improve NK cell activity in patients with cancer and call for more preclinical assessments of the potential of IMIDs in NK cell activation.

[Neonatal immunology and cord blood transplantation]. / Immunologie néonatale et greffe de sang de cordon.

The increased susceptibility of human newborns to infections is usually ascribed to the immaturity of the neonatal immune system. The neonatal immune system has never met microbial antigens, and thus the repertoire of its adaptative arm (T and B cells) is entirely pre-immune, or "naïve". However this neonatal pre-immune repertoire is similar to the adult pre-immune repertoire, and cord blood natural killer cells studies show that the innate immunity cells harbor the full killing machinery that characterize mature cells. Moreover, human neonates are able to show an adult-like allogeneic response. Taken together, several lines of evidence suggest that the neonatal immune system, although naïve, is fully mature. However, newborns display phenotypic and functional differences with adults in both adaptative and innate arms. Specific properties may explain these differences, as high number of regulatory T cells, low plasmacytoid dendritic cell response to stimuli and high IL-10 production. These properties are in line with the high susceptibility of newborns to infections and the low incidence of graft-versus-host-disease after cord blood transplantation. To explain these differences, we introduce a new model. Although naive, the neonatal immune system is mature, and these functional differences are due to a message originating from the placenta and aimed at inducing the foetus tolerance to its mother. Full understanding of the involved mechanisms will help to protect the newborn against infections and to improve cord blood transplantation outcome.

Late-onset hemophagocytic lymphohistiocytosis with neurological presentation.

Missense mutations in genes involved in familial hemophagocytic lymphohistiocytosis can delay the onset of this life-threatening disease. In children and adults, early recognition of aspecific features as neurological symptoms is crucial as urgent treatment is required.

TGF-ß inhibits the activation and functions of NK cells by repressing the mTOR pathway.

Transforming growth factor-ß (TGF-ß) is a major immunosuppressive cytokine that maintains immune homeostasis and prevents autoimmunity through its antiproliferative and anti-inflammatory properties in various immune cell types. We provide genetic, pharmacologic, and biochemical evidence that a critical target of TGF-ß signaling in mouse and human natural killer (NK) cells is the serine and threonine kinase mTOR (mammalian target of rapamycin). Treatment of mouse or human NK cells with TGF-ß in vitro blocked interleukin-15 (IL-15)-induced activation of mTOR. TGF-ß and the mTOR inhibitor rapamycin both reduced the metabolic activity and proliferation of NK cells and reduced the abundances of various NK cell receptors and the cytotoxic activity of NK cells. In vivo, constitutive TGF-ß signaling or depletion of mTOR arrested NK cell development, whereas deletion of the TGF-ß receptor subunit TGF-ßRII enhanced mTOR activity and the cytotoxic activity of the NK cells in response to IL-15. Suppression of TGF-ß signaling in NK cells did not affect either NK cell development or homeostasis; however, it enhanced the ability of NK cells to limit metastases in two different tumor models in mice. Together, these results suggest that the kinase mTOR is a crucial signaling integrator of pro- and anti-inflammatory cytokines in NK cells. Moreover, we propose that boosting the metabolic activity of antitumor lymphocytes could be an effective strategy to promote immune-mediated tumor suppression.

T-bet and Eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow.

Trail(+)DX5(-)Eomes(-) natural killer (NK) cells arise in the mouse fetal liver and persist in the adult liver. Their relationships with Trail(-)DX5(+) NK cells remain controversial. We generated a novel Eomes-GFP reporter murine model to address this question. We found that Eomes(-) NK cells are not precursors of classical Eomes(+) NK cells but rather constitute a distinct lineage of innate lymphoid cells. Eomes(-) NK cells are strictly dependent on both T-bet and IL-15, similarly to NKT cells. We observed that, in the liver, expression of T-bet in progenitors represses Eomes expression and the development of Eomes(+) NK cells. Reciprocally, the bone marrow (BM) microenvironment restricts T-bet expression in developing NK cells. Ectopic expression of T-bet forces the development of Eomes(-) NK cells, demonstrating that repression of T-bet is essential for the development of Eomes(+) NK cells. Gene profile analyses show that Eomes(-) NK cells share part of their transcriptional program with NKT cells, including genes involved in liver homing and NK cell receptors. Moreover, Eomes(-) NK cells produce a broad range of cytokines, including IL-2 and TNF in vitro and in vivo, during immune responses against vaccinia virus. Thus, mutually exclusive expression of T-bet and Eomes drives the development of different NK cell lineages with complementary functions.