Pregnenolone sulfate induces transcriptional and immunoregulatory effects on T cells.

Pregnenolone sulfate is a steroid metabolite of the steroidogenesis precursor, pregnenolone, with similar functional properties, including immunosuppression. We recently reported an elevation in serum levels of pregnenolone sulfate in children with malaria, contributing to an immunosuppressed state. Yet, the molecular mechanisms in which this steroid exerts its immunoregulatory functions are lacking. In this study, we examined the effects of pregnenolone sulfate on T cell viability, proliferation and transcriptome. We observed a pregnenolone sulfate dose-dependent induction of T cell death and reduction in proliferation. RNA sequencing analysis of pregnenolone sulfate-treated T cells for 2 and 24 h revealed the downregulation of pro-inflammatory genes and the upregulation of the steroid nuclear receptor superfamily, NR4A, as early-response genes. We also report a strong activation of the integrated stress response mediated by the upregulation of EIF2AK3. These results contribute to the knowledge on transcriptional regulation driving the immunoregulatory effects of pregnenolone sulfate on T cells.

Impact of Plasmodium falciparum infection on DNA methylation of circulating immune cells.

The regulation of immune cell responses to infection is a complex process that involves various molecular mechanisms, including epigenetic regulation. DNA methylation has been shown to play central roles in regulating gene expression and modulating cell response during infection. However, the nature and extent to which DNA methylation is involved in the host immune response in human malaria remains largely unknown. Here, we present a longitudinal study investigating the temporal dynamics of genome-wide in vivo DNA methylation profiles using 189 MethylationEPIC 850 K profiles from 66 children in Burkina Faso, West Africa, sampled three times: before infection, during symptomatic parasitemia, and after malaria treatment. The results revealed major changes in the DNA methylation profiles of children in response to both Plasmodium falciparum infection and malaria treatment, with widespread hypomethylation of CpGs upon infection (82% of 6.8 K differentially methylated regions). We document a remarkable reversal of CpG methylation profiles upon treatment to pre-infection states. These changes implicate divergence in core immune processes, including the regulation of lymphocyte, neutrophil, and myeloid leukocyte function. Integrative DNA methylation-mRNA analysis of a top differentially methylated region overlapping the pro-inflammatory gene TNF implicates DNA methylation of TNF cis regulatory elements in the molecular mechanisms of TNF regulation in human malaria. Our results highlight a central role of epigenetic regulation in mounting the host immune response to P. falciparum infection and in response to malaria treatment.

NMR-Based Analysis of Nanobodies to SARS-CoV-2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID-19.

Following the entry into the host cell, SARS-CoV-2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B-cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID-19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild-type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication.

Metabolome modulation of the host adaptive immunity in human malaria.

Host responses to infection with the malaria parasite Plasmodium falciparum vary among individuals for reasons that are poorly understood. Here we reveal metabolic perturbations as a consequence of malaria infection in children and identify an immunosuppressive role of endogenous steroid production in the context of P. falciparum infection. We perform metabolomics on matched samples from children from two ethnic groups in West Africa, before and after infection with seasonal malaria. Analysing 306 global metabolomes, we identify 92 parasitaemia-associated metabolites with impact on the host adaptive immune response. Integrative metabolomic and transcriptomic analyses, and causal mediation and moderation analyses, reveal an infection-driven immunosuppressive role of parasitaemia-associated pregnenolone steroids on lymphocyte function and the expression of key immunoregulatory lymphocyte genes in the Gouin ethnic group. In children from the less malaria-susceptible Fulani ethnic group, we observe opposing responses following infection, consistent with the immunosuppressive role of endogenous steroids in malaria. These findings advance our understanding of P. falciparum pathogenesis in humans and identify potential new targets for antimalarial therapeutic interventions.

Integrative genomic analysis reveals mechanisms of immune evasion in P. falciparum malaria.

The mechanisms behind the ability of Plasmodium falciparum to evade host immune system are poorly understood and are a major roadblock in achieving malaria elimination. Here, we use integrative genomic profiling and a longitudinal pediatric cohort in Burkina Faso to demonstrate the role of post-transcriptional regulation in host immune response in malaria. We report a strong signature of miRNA expression differentiation associated with P. falciparum infection (127 out of 320 miRNAs, B-H FDR 5%) and parasitemia (72 miRNAs, B-H FDR 5%). Integrative miRNA-mRNA analysis implicates several infection-responsive miRNAs (e.g., miR-16-5p, miR-15a-5p and miR-181c-5p) promoting lymphocyte cell death. miRNA cis-eQTL analysis using whole-genome sequencing data identified 1,376 genetic variants associated with the expression of 34 miRNAs (B-H FDR 5%). We report a protective effect of rs114136945 minor allele on parasitemia mediated through miR-598-3p expression. These results highlight the impact of post-transcriptional regulation, immune cell death processes and host genetic regulatory control in malaria.

Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming.

Human mesenchymal stromal cells (MSC) have been shown to dampen immune response and promote tissue repair, but the underlying mechanisms are still under investigation. Herein, we demonstrate that umbilical cord-derived MSC (UC-MSC) alter the phenotype and function of monocyte-derived dendritic cells (DC) through lactate-mediated metabolic reprogramming. UC-MSC can secrete large quantities of lactate and, when present during monocyte-to-DC differentiation, induce instead the acquisition of M2-macrophage features in terms of morphology, surface markers, migratory properties and antigen presentation capacity. Microarray expression profiling indicates that UC-MSC modify the expression of metabolic-related genes and induce a M2-macrophage expression signature. Importantly, monocyte-derived DC obtained in presence of UC-MSC, polarize naïve allogeneic CD4+ T-cells into Th2 cells. Treatment of UC-MSC with an inhibitor of lactate dehydrogenase strongly decreases lactate concentration in culture supernatant and abrogates the effect on monocyte-to-DC differentiation. Metabolic analysis further revealed that UC-MSC decrease oxidative phosphorylation in differentiating monocytes while strongly increasing the spare respiratory capacity proportional to the amount of secreted lactate. Because both MSC and monocytes are recruited in vivo at the site of tissue damage and inflammation, we propose the local increase of lactate concentration induced by UC-MSC and the consequent enrichment in M2-macrophage generation as a mechanism to achieve immunomodulation.

Role of Natural Killer Cells in Intravenous Immunoglobulin-Induced Graft-versus-Host Disease Inhibition in NOD/LtSz-scidIL2rg(-/-) (NSG) Mice.

Although clinical studies have yet to demonstrate clearly the use of intravenous immunoglobulin (IVIG) for prevention of graft-versus-host disease (GVHD), their effective use in a xenogeneic mouse model has been demonstrated. We aimed to determine the mechanism of action by which IVIG contributes to GVHD prevention in a xenogeneic mouse model. NOD/LtSz-scidIL2rg(-/-) (NSG) mice were used for our xenogeneic mouse model of GVHD. Sublethally irradiated NSG mice were injected with human peripheral blood mononuclear cells (huPBMCs) and treated weekly with PBS or 50 mg IVIG. Incidence of GVHD and survival were noted, along with analysis of cell subsets proliferation in the peripheral blood. Weekly IVIG treatment resulted in a robust and consistent proliferation of human natural killer cells that were activated, as demonstrated by their cytotoxicity against K562 target cells. IVIG treatment did not inhibit GVHD when huPBMCs were depleted in natural killer (NK) cells, strongly suggesting that this NK cell expansion was required for the IVIG-mediated prevention of GVHD in our mouse model. Moreover, inhibition of T cell activation by either cyclosporine A (CsA) or monoclonal antihuman CD3 antibodies abolished the IVIG-induced NK cell expansion. In conclusion, IVIG treatment induces NK cell proliferation, which is essential for IVIG-mediated protection of GVHD in our mouse model. Furthermore, activated T cells are mandatory for effective IVIG-induced NK cell proliferation. These results shed light on a new mechanism of action of IVIG and could explain why the efficacy of IVIG in preventing GVHD in a clinical setting, where patients receive CsA, has never been undoubtedly demonstrated.

Implication of different effector mechanisms by cord blood-derived and peripheral blood-derived cytokine-induced killer cells to kill precursor B acute lymphoblastic leukemia cell lines.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cells ex vivo-expanded from cord blood (CB) or peripheral blood (PB) have been shown to be cytotoxic against autologous and allogeneic tumor cells. We have previously shown that CD56(+) CIK cells (CD3(+)CD56(+) and CD3(-)CD56(+)) are capable of killing precursor B-cell acute lymphoblastic leukemia (B-ALL) cell lines. However, the lytic pathways used by CD56(+) PB and CB-CIK cells to kill B-ALL cell lines have not been studied. METHODS: CB and PB-CIK cells were differentiated. CD56(+) CB- and PB-CIK cells were compared for expression of different phenotypic markers and for the lytic pathways used to kill B-ALL cell lines. RESULTS: We found that cytotoxic granule proteins were expressed at higher levels in CD56(+) PB-CIK than in CD56(+) CB-CIK cells. However, CD56(+) CB-CIK cells expressed more tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) compared with CD56(+) PB-CIK cells. We observed that CD56(+) CB-CIK cells used both the NKG2D and TRAIL cytotoxic pathways and were more effective at killing REH cells than CD56(+) PB-CIK cells that used only the NKG2D pathway. In contrast, CD56(+) PB-CIK cells used both NKG2D and TRAIL pathways to kill NALM6 cells, whereas CD56(+) CB-CIK cells used only the NKG2D pathway. CONCLUSIONS: Our results suggest that both the source of CIK and the type of B-ALL cell line have an impact on the intensity of the cytolytic activity and on the pathway used. These findings may have clinical implications with respect to optimizing therapeutic efficacy, which may be dependent on the source of the CIK cells and on the target tumor cells.

Efficient BST2 antagonism by Vpu is critical for early HIV-1 dissemination in humanized mice.

BACKGROUND: Vpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(ß-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions. RESULTS: Using a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the ß-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to ß-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated ß-TrCP-dependent BST-2 degradation for optimal initial viral propagation. CONCLUSIONS: Overall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

Cord blood-derived and peripheral blood-derived cytokine-induced killer cells are sensitive to Fas-mediated apoptosis.

Fas-mediated apoptosis is one of the mechanisms used by tumor cells to escape the cytotoxicity of tumor-infiltrating lymphocytes. It has been suggested that cytokine-induced killer (CIK) cells are resistant to Fas-mediated apoptosis, thereby rendering them more attractive for use in cellular immunotherapy. Unlike what was observed by others, here we show that CIK cells are sensitive to Fas-mediated apoptosis. We have observed an increase in Fas expression in the different CIK cell subpopulations (CD3(+)CD56(-), CD3(+)CD56(+), and CD3(-)CD56(+)) isolated from both cord blood (CB) and peripheral blood (PB). We also show that the bulk, as well as the CD3(+)CD56(-) and CD56(+) CB- and PB-CIK cell subpopulations were sensitive to Fas-mediated apoptosis induced by both CH11 and APO-1 antibodies, albeit with a weaker effect for the CH11 antibody on CB-CIK cells. In addition, in the presence of the APO-1 and CH11 inducers, Fas engagement inhibited the cytotoxic activity of CB- and PB-CIK cells. This new contradictory result may help explain the variable efficacy observed with CIK cells in the clinic.

Cord-blood-derived mesenchymal stromal cells downmodulate CD4+ T-cell activation by inducing IL-10-producing Th1 cells.

The mechanisms by which mesenchymal stromal cells (MSCs) induce immunomodulation are still poorly understood. In the current work, we show by a combination of polymerase chain reaction (PCR) array, flow cytometry, and multiplex cytokine data analysis that during the inhibition of an alloantigen-driven CD4+ T-cell response, MSCs induce a fraction of CD4+ T-cells to coexpress interferon-γ (IFNγ) and interleukin-10 (IL-10). This CD4+ IFNγ+ IL-10+ cell population shares properties with recently described T-cells originating from switched Th1 cells that start producing IL-10 and acquire a regulatory function. Here we report that IL-10-producing Th1 cells accumulated with time during T-cell stimulation in the presence of MSCs. Moreover, MSCs caused stimulated T-cells to downregulate the IFNγ receptor (IFNγR) without affecting IL-10 receptor expression. Further, the inhibitory effect of MSCs could be reversed by an anti-IFNγR-blocking antibody, indicating that IFNγ is one of the major players in MSC-induced T-cell suppression. Stimulated (and, to a lesser extent, resting) CD4+ T-cells treated with MSCs were able to inhibit the proliferation of autologous CD4+ T-cells, demonstrating their acquired regulatory properties. Altogether, our results suggest that the generation of IL-10-producing Th1 cells is one of the mechanisms by which MSCs can downmodulate an immune response.

Human interferon-alpha increases the cytotoxic effect of CD56(+) cord blood-derived cytokine-induced killer cells on human B-acute lymphoblastic leukemia cell lines.

BACKGROUND AIMS: Cytokine-induced killer (CIK) cells may represent a promising immunotherapy for the treatment of children with relapsing B-lineage acute lymphoblastic leukemia (B-ALL) following hematopoietic stem cell transplantation (HSCT). Therefore, we investigated the possibility of combining adoptive immunotherapy with CIK cells and human interferon-alpha (hIFN-α) in order to potentiate the cytotoxicity of CIK cells against B-ALL. METHODS: Cord blood- derived CIK (CB-CIK) cells were differentiated, stimulated with phosphate-buffered saline (PBS) or hIFN-α, and tested for cytotoxic activity. We tested the anti-leukemic and graft-versus-host disease (GvHD) effects of CB-CIK cells in a human xenograft NOD/SCID/γc(-) (NSG) mouse model. RESULTS: Bulk CB-CIK cells showed very moderate cytotoxic activity while the subpopulation of CD56(+) CB-CIK cells showed significant cytotoxic activity against B-ALL cells. hIFN-α significantly augmented the cytotoxicity of CD56(+)CB-CIK cells in vitro and induced signal transducer and activator of transcription-1 (STAT1) phosphorylation. In addition, CD56(+)CB-CIK cells could delay mouse mortality significantly in vivo, and this effect was enhanced significantly by hIFN-α (P = 0.022). Furthermore, unlike CB mononuclear cells or peripheral blood mononuclear cells (PBMC), CD56(+)CB-CIK cells, alone or stimulated with hIFN-α, caused either no GvHD or mild GvHD, respectively, when injected into sublethally irradiated NSG mice. CONCLUSIONS: CD56(+)CB-CIK cells are effective cytotoxic agents against human B-ALL cell lines in vitro and possess anti-leukemic activity that is potentiated by hIFN-α in an NSG mouse model in vivo. These pre-clinical data support the testing of this immunotherapeutic approach in the clinic for the treatment of B-ALL.

Therapeutic efficacy of cord blood-derived mesenchymal stromal cells for the prevention of acute graft-versus-host disease in a xenogenic mouse model.

Human mesenchymal stromal cells (MSCs) have been successfully utilized for the treatment of refractory graft-versus-host disease (GvHD). Despite the large number of in vitro and in vivo models developed for clarifying their immunomodulatory properties, the mechanism of action of MSCs remains elusive and their efficacy controversial. Here, we tested the ability of cord blood-derived MSCs to alleviate the symptoms of GvHD induced by the injection of human peripheral blood mononuclear cells into NOD/SCID/γc(-) mice. In this in vivo xeno-GvHD model, we demonstrate that a single MSC injection is able to inhibit GvHD in terms of clinical signs and related mortality. We also show that in this model MSCs act by both immunomodulating T-cells and fostering recovery after irradiation. The translational impact of these findings could provide a reliable preclinical model for studying the efficacy, dosage, and time of administration of human MSCs for the prevention of acute GvHD.