Multimeric immunotherapeutic complexes activating natural killer cells towards HIV-1 cure.

BACKGROUND: Combination antiretroviral therapy (cART) has dramatically extended the life expectancy of people living with HIV-1 and improved their quality of life. There is nevertheless no cure for HIV-1 infection since HIV-1 persists in viral reservoirs of latently infected CD4+ T cells. cART does not eradicate HIV-1 reservoirs or restore cytotoxic natural killer (NK) cells which are dramatically reduced by HIV-1 infection, and express the checkpoint inhibitors NKG2A or KIR2DL upregulated after HIV-1 infection. Cytotoxic NK cells expressing the homing receptor CXCR5 were recently described as key subsets controlling viral replication. METHODS: We designed and evaluated the potency of "Natural killer activating Multimeric immunotherapeutic compleXes", called as NaMiX, combining multimers of the IL-15/IL-15Rα complex with an anti-NKG2A or an anti-KIR single-chain fragment variable (scFv) to kill HIV-1 infected CD4+ T cells. The oligomerization domain of the C4 binding protein was used to associate the IL-15/IL-15Rα complex to the scFv of each checkpoint inhibitor as well as to multimerize each entity into a heptamer (α form) or a dimer (ß form). Each α or ß form was compared in different in vitro models using one-way ANOVA and post-hoc Tukey's tests before evaluation in humanized NSG tg-huIL-15 mice having functional NK cells. RESULTS: All NaMiX significantly enhanced the cytolytic activity of NK and CD8+ T cells against Raji tumour cells and HIV-1+ ACH-2 cells by increasing degranulation, release of granzyme B, perforin and IFN-γ. Targeting NKG2A had a stronger effect than targeting KIR2DL due to higher expression of NKG2A on NK cells. In viral inhibition assays, NaMiX initially increased viral replication of CD4+ T cells which was subsequently inhibited by cytotoxic NK cells. Importantly, anti-NKG2A NaMiX enhanced activation, cytotoxicity, IFN-γ production and CXCR5 expression of NK cells from HIV-1 positive individuals. In humanized NSG tg-huIL-15 mice, we confirmed enhanced activation, degranulation, cytotoxicity of NK cells, and killing of HIV-1 infected cells from mice injected with the anti-NKG2A.α NaMiX, as compared to control mice, as well as decreased total HIV-1 DNA in the lung. CONCLUSIONS: NK cell-mediated killing of HIV-1 infected cells by NaMiX represents a promising approach to support HIV-1 cure strategies.

Push-Through Filtration of Emulsified Adipose Tissue Over a 500-µm Mesh Significantly Reduces the Amount of Stromal Vascular Fraction and Mesenchymal Stem Cells.

BACKGROUND: Mechanical isolation of the stromal vascular fraction (SVF) separates the stromal component from the parenchymal cells. Emulsification is currently the most commonly used disaggregation method and is effective in disrupting adipocytes and fragmenting the extracellular matrix (ECM). Subsequent push-through filtration of emulsified adipose tissue removes parts of the ECM that are not sufficiently micronized, thereby further liquifying the tissue. OBJECTIVES: The aim of this study was to investigate whether filtration over a 500-µm mesh filter might affect the SVF and adipose-derived mesenchymal stem cell (MSC) quantity in emulsified lipoaspirate samples by removing ECM fragments. METHODS: Eleven lipoaspirate samples from healthy nonobese women were harvested and emulsified in 30 passes. One-half of the sample was filtered through a 500-µm mesh filter and the other half was left unfiltered. Paired samples were processed and analyzed by flow cytometry to identify cellular viability, and SVF and MSC yield. RESULTS: Push-through filtration reduced the number of SVF cells by a mean [standard deviation] of 39.65% [5.67%] (P < .01). It also significantly reduced MSC counts by 48.28% [6.72%] (P < .01). Filtration did not significantly affect viability (P = .118). CONCLUSIONS: Retention of fibrous remnants by push-through filters removed ECM containing the SVF and MSCs from emulsified lipoaspirates. Processing methods should aim either to further micronize the lipoaspirate before filtering or not to filter the samples at all, to preserve both the cellular component carried within the ECM and the inductive properties of the ECM itself.

Autologous dendritic cell vaccination against HIV-1 induces changes in natural killer cell phenotype and functionality.

Although natural killer (NK) cells have been studied in connection with dendritic cell (DC)-based vaccination in the field of cancer immunology, their role has barely been addressed in the context of therapeutic vaccination against HIV-1. In this study, we evaluated whether a therapeutic DC-based vaccine consisting of monocyte-derived DCs electroporated with Tat, Rev and Nef encoding mRNA affects NK cell frequency, phenotype and functionality in HIV-1-infected individuals. Although the frequency of total NK cells did not change, we observed a significant increase in cytotoxic NK cells following immunisation. In addition, significant changes in the NK cell phenotype associated with migration and exhaustion were observed together with increased NK cell-mediated killing and (poly)functionality. Our results show that DC-based vaccination has profound effects on NK cells, which highlights the importance of evaluating NK cells in future clinical trials looking at DC-based immunotherapy in the context of HIV-1 infection.

Qualitative plasma viral load determination as a tool for screening of viral reservoir size in PWH.

OBJECTIVES: Suppression of viral replication in patients on antiretroviral therapy (ART) is determined by plasma viral load (pVL) measurement. Whenever pVL reaches values below the limit of quantification, the qualitative parameter 'target detected' or 'target not detected' is available but often not reported to the clinician. We investigated whether qualitative pVL measurements can be used to estimate the viral reservoir size. DESIGN: The study recruited 114 people with HIV (PWH) who are stable on ART between 2016 and 2018. The percentage of pVL measurements qualitatively reported as 'target detected' (PTD) within a 2-year period was calculated. METHODS: t-DNA and US-RNA were used to estimate viral reservoir size and were quantified on peripheral blood mononuclear cells (PBMCs) using droplet digital PCR. RESULTS: A median of 6.5 pVL measurements over a 2-year period was evaluated for each participant to calculate PTD. A positive correlation was found between t-DNA and PTD (r = 0.24; P = 0.011) but not between US-RNA and PTD (r = 0.1; P = 0.3). A significantly lower PTD was observed in PWH with a small viral reservoir, as estimated by t-DNA less than 66 copies/106 PBMCs and US-RNA less than 10 copies/106 PBMCs, compared with PWH with a larger viral reservoir (P = 0.001). We also show that t-DNA is detectable whenever PTD is higher than 56% and that ART regimen does not affect PTD. CONCLUSION: Our study shows that PTD provides an efficient parameter to preselect participants with a small viral reservoir based on already available pVL data for future HIV cure trials.

Efficient Induction of Antigen-Specific CD8+ T-Cell Responses by Cationic Peptide-Based mRNA Nanoparticles.

A major determinant for the success of mRNA-based vaccines is the composition of the nanoparticles (NPs) used for formulation and delivery. Cationic peptides represent interesting candidate carriers for mRNA, since they have been shown to efficiently deliver nucleic acids to eukaryotic cells. mRNA NPs based on arginine-rich peptides have previously been demonstrated to induce potent antigen-specific CD8+ T-cell responses. We therefore compared the histidine-rich amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA) to the fully substituted arginine variant (LAH4-L1R) for their capacity to formulate mRNA and transfect dendritic cells (DCs). Although both peptides encapsulated mRNA to the same extent, and showed excellent uptake in DCs, the gene expression level was significantly higher for LAH4-L1. The LAH4-L1-mRNA NPs also resulted in enhanced antigen presentation in the context of MHC I compared to LAH4-L1R in primary murine CD103+ DCs. Both peptides induced DC maturation and inflammasome activation. Subsequent ex vivo stimulation of OT-I splenocytes with transfected CD103+ DCs resulted in a high proportion of polyfunctional CD8+ T cells for both peptides. In addition, in vivo immunization with LAH4-L1 or LAH4-L1R-mRNA NPs resulted in proliferation of antigen-specific T cells. In conclusion, although LAH4-L1 outperformed LAH4-L1R in terms of transfection efficiency, the immune stimulation ex vivo and in vivo was equally efficient.

Oncolytic Herpes Simplex Virus Type 1 Induces Immunogenic Cell Death Resulting in Maturation of BDCA-1+ Myeloid Dendritic Cells.

Recently, a paradigm shift has been established for oncolytic viruses (OVs) as it was shown that the immune system plays an important role in the specific killing of tumor cells by OVs. OVs have the intrinsic capacity to provide the right signals to trigger anti-tumor immune responses, on the one hand by delivering virus-derived innate signals and on the other hand by inducing immunogenic cell death (ICD), which is accompanied by the release of various damage-associated molecules from infected tumor cells. Here, we determined the ICD-inducing capacity of Talimogene laherparepvec (T-VEC), a herpes simplex virus type 1 based OV, and benchmarked this to other previously described ICD (e.g., doxorubicin) and non-ICD inducing agents (cisplatin). Furthermore, we studied the capability of T-VEC to induce the maturation of human BDCA-1+ myeloid dendritic cells (myDCs). We found that T-VEC treatment exerts direct and indirect anti-tumor effects as it induces tumor cell death that coincides with the release of hallmark mediators of ICD, while simultaneously contributing to the maturation of BDCA-1+ myDCs. These results unequivocally cement OVs in the category of cancer immunotherapy.

Butyrate Does Not Protect Against Inflammation-induced Loss of Epithelial Barrier Function and Cytokine Production in Primary Cell Monolayers From Patients With Ulcerative Colitis.

BACKGROUND AND AIMS: In vitro studies using immortalised cancer cell lines showed that butyrate has an overall positive effect on epithelial barrier integrity, but the physiological relevance of cancer cell lines is limited. We developed epithelial monolayers from human tissue samples of patients with ulcerative colitis [UC] to assess the effect of butyrate on epithelial barrier function. METHODS: A protocol to establish monolayers from primary epithelial cells of UC patients [n = 10] and non-UC controls [n = 10] was optimised. The monolayers were treated with 8 mM sodium butyrate ± tumour necrosis factor alpha [TNFα] and type II interferon [IFNγ] for 48 h. Changes in transepithelial electrical resistance were monitored. Barrier gene expression levels were measured. Inflammatory proteins in the supernatant of the cells were quantified with OLINK. RESULTS: We demonstrated that primary monolayer cultures can be grown within 1 week of culture with robust resistance values and polarised tight junction expression. Butyrate treatment of the cultures increased resistance but was detrimental in combination with TNFα and IFNγ. The combined treatment further induced even higher IL8 mRNA and inflammatory protein secretion than for the inflammatory mediators alone. The observed effects were similar in cultures from patients and non-UC controls, suggesting that there were no patient-specific responses responsible for these findings. CONCLUSIONS: We found that butyrate does not protect against inflammation-induced barrier dysfunction and even worsens its effects in primary epithelial monolayers of UC patients and controls. The basic mechanisms of butyrate should therefore be reconsidered in future studies, in particular in patients with active inflammation and pre-existing barrier defects as is known for UC.

Signal transduction analysis of the NLRP3-inflammasome pathway after cellular damage and its paracrine regulation.

Activation of the NLRP3-inflammasome pathway and production of the inflammatory cytokine IL-1B after cellular damage caused by infarct or infection is a key process in several diseases such as acute myocardial infarction and inflammatory bowel disease. However, while the molecular triggers of the NLRP3-pathway after cellular damage are well known, the mechanisms that sustain or confine its activity are currently under investigation. We present here an Ordinary Differential Equation-based model that investigates the mechanisms of inflammasome activation and regulation in monocytes to predict IL-1ß activation kinetics upon a two-step activation by Damage-Associate-Molecular-Particles (DAMP) and extracellular ATP. Assuming both activation signals to be concomitantly present or present with a delay of 12h, the model predicted a transient IL-1ß activation at different concentration levels dependent on signal synchronisation. Introducing a positive feedback loop mediated by active IL-1ß resulted in a sustained IL-1ß activation, hence arguing for a paracrine signalling between inflammatory cells to guarantee a temporally stable inflammatory response. We then investigate mechanisms that control termination of inflammation using two recently identified molecular intervention points in the inflammasome pathway. We found that a more upstream regulation, by attenuating production of the IL-1ß-proform, was more potent in attenuating active IL-1ß production than direct inhibition of the NLRP3-inflammasome. Interestingly, ablating this upstream negative feedback led to a high variability of IL-1ß production in monocytes from different subjects, consistent with a recent pre-clinical study. We finally discuss the relevance and implications of our findings in disease models of acute myocardial infarction and spontaneous colitis.