Human CD4 Research Articles

In vivo affinity maturation of the CD4 domains of an HIV-1-entry inhibitor.

Human proteins repurposed as biologics for clinical use have been engineered through in vitro techniques that improve the affinity of the biologics for their ligands. However, the techniques do not select against properties, such as protease sensitivity or self-reactivity, that impair the biologics' clinical efficacy. Here we show that the B-cell receptors of primary murine B cells can be engineered to affinity mature in vivo the human CD4 domains of the HIV-1-entry inhibitor CD4 immunoadhesin (CD4-Ig). Specifically, we introduced genes encoding the CD4 domains 1 and 2 (D1D2) of a half-life-enhanced form of CD4-Ig (CD4-Ig-v0) into the heavy-chain loci of murine B cells and adoptively transferred these cells into wild-type mice. After immunization, the B cells proliferated, class switched, affinity matured and produced D1D2-presenting antibodies. Somatic hypermutations in the D1D2-encoding region of the engrafted cells improved the binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein and the inhibitor's ability to neutralize a panel of HIV-1 isolates without impairing its pharmacokinetic properties. In vivo affinity maturation of non-antibody protein biologics may guide the development of more effective therapeutics.

The identification of a SARs-CoV2 S2 protein-derived peptide with super-antigen-like stimulatory properties on T-cells.

Severe COVID-19 can trigger a cytokine storm, leading to acute respiratory distress syndrome (ARDS) with similarities to superantigen-induced toxic shock syndrome. An outstanding question is whether SARS-CoV-2 protein sequences can directly induce inflammatory responses. In this study, we identify a region in the SARS-CoV-2 S2 spike protein with sequence homology to bacterial super-antigens (termed P3). Computational modeling predicts P3 binding to sites on MHC class I/II and the TCR that partially overlap with sites for the binding of staphylococcal enterotoxins B and H. Like SEB and SEH peptides, P3 stimulated 25-40% of human CD4+ and CD8+ T cells, increasing IFN-γ and granzyme B production. viSNE and SPADE profiling identified overlapping and distinct IFN-γ and GZMB subsets. The super-antigenic properties of P3 were further evident by its selective expansion of T cells expressing specific TCR Vα and Vβ chain repertoires. In vivo experiments in mice revealed that the administration of P3 led to a significant upregulation of proinflammatory cytokines IL-1β, IL-6, and TNF-α. While the clinical significance of P3 in COVID-19 remains unclear, its homology to other mammalian proteins suggests a potential role for this peptide family in human inflammation and autoimmunity.

Development of immunocompetent full thickness skin tissue constructs to model skin fibrosis for high-throughput drug screening.

The lack of the immune component in most of the engineered skin models remains a challenge to study the interplay between different immune and non-immune cell types of the skin. Immunocompetent human in vitro skin models offer potential advantages in recapitulating in vivo like behavior which can serve to accelerate translational research and therapeutics development for skin diseases. Here we describe a 3D human full-thickness skin (FTS) equivalent incorporating polarized M1 and M2 macrophages from human peripheral CD14+ monocytes. This macrophage-incorporated FTS model demonstrates discernible immune responses with physiologically relevant cytokine production and macrophage plasticity under homeostatic and lipopolysaccharide stimulation conditions. M2-incorporated FTS recapitulates skin fibrosis phenotypes with transforming growth factor-β1 treatment as reflected by significant collagen deposition and myofibroblast expression, demonstrating a M2 potentiation effect. In conclusion, we successfully biofabricated an immunocompetent FTS with functional macrophages in a high-throughput (HT) amenable format. This model is the first step towards a HT-assay platform to develop new therapeutics for skin diseases.&#xD.

Inborn errors of immunity reveal the molecular requirements for the generation and maintenance of human IL-9 expressing cells.

CD4+ T cells play essential roles in adaptive immunity. Distinct CD4+ T cell subsets - Th1, Th2, Th17, Th22, T follicular helper and regulatory T cells - have been identified and their contributions to host defence and immune regulation are increasingly well-defined. IL-9 producing Th9 cells were first described in 2008 and appear to play both protective and pathogenic roles in human immunity. However, key requirements for generating human Th9 cells remain incompletely defined. Define signalling pathways that regulate IL-9 production by human CD4+ T cells. Human naive and memory CD4+ T cells were cultured under different conditions and the molecular mechanisms regulating IL-9 induction were determined by assessing the ability of CD4+ T cells from a broad range of patients (n=92) with pathogenic variants in key immune genes (n=21) to differentiate into IL-9+ cells. We identified two culture conditions that yielded IL-9-expressing cells from naïve CD4+ T cells, and amplified IL-9 production by in vivo-generated memory CD4+ T cells: TGFβ plus IL-4 (i.e. Th9 polarising condition), and the combination of IL-21, IL-23, IL-6, IL-1β and TGFβ (i.e. Th17 polarising condition). Combining these conditions had a synergistic effect in generating IL-9+CD4+ T cells. IL-9 induction required STAT3-activating cytokines as well as intact signalling via the TCR and STAT5. Importantly, IL-9 induction was restrained by IFNγ/STAT1 and IL-10. Our findings revealed critical molecules involved in inducing/restraining IL-9 production by human CD4+ T cells, thereby identifying pathways that could be targeted to modulate IL-9 in health and disease.

In human CD4+ T-Cells, omeprazole suppresses proliferation, downregulates V-ATPase, and promotes differentiation toward an autoimmunity-favoring phenotype

BackgroundProton pump inhibitors (PPIs) represent a commonly prescribed class of medications. Triggered by findings indicating a correlation between PPI usage and susceptibility to infectious or autoimmune diseases, we studied the impact of a pharmacological concentration of omeprazole on human CD4+ T-cells. MethodsIn mixed lymphocyte reactions (MLRs), we analyzed the proliferation index and measured the concentration of key cytokines representative of distinct CD4+ T-cell subsets. In CD4+ T-cells isolated from the MLRs, we evaluated proliferation markers and pathways, the expression of signature transcription factors of CD4+ T-cell subsets, vacuolar H+- ATPase (V-ATPase) levels, and the activation status of AMP-activated kinase (AMPK) and mammalian target of rapamycin complex-1 (mTORC1). ResultsOmeprazole reduced proliferation index in MLRs, and in isolated CD4+ T-cells, it downregulated the proliferation marker Ki-67, possibly mediated by the p53- p21 pathway. Analysis of cytokines and signature transcription factors of CD4+ T-cell subsets indicated that omeprazole decreased T helper 1 (Th1) differentiation, had negligible impact on Th2 differentiation, increased Th17 differentiation, and reduced regulatory T-cell (Treg) differentiation. Omeprazole also decreased V-ATPase, a known target of PPIs and a site for AMPK and mTORC1 activation. Consequently, this led to diminished activation of these kinases, potentially elucidating the mechanism by which omeprazole influences CD4+ T-cell differentiation. ConclusionOmeprazole downregulates V-ATPase and inhibits activation of AMPK and mTORC1. As a result, omeprazole suppresses CD4+ T-cell clonal expansion, potentially contributing to the observed association between PPIs and susceptibility to infections. Additionally, it modulates CD4+ T-cell differentiation in a manner that favors autoimmunity.

Integrative mapping of human CD8+ T cells in inflammation and cancer.

CD8+ T cells exhibit remarkable phenotypic diversity in inflammation and cancer. However, a comprehensive understanding of their clonal landscape and dynamics remains elusive. Here we introduce scAtlasVAE, a deep-learning-based model for the integration of large-scale single-cell RNA sequencing data and cross-atlas comparisons. scAtlasVAE has enabled us to construct an extensive human CD8+ T cell atlas, comprising 1,151,678 cells from 961 samples across 68 studies and 42 disease conditions, with paired T cell receptor information. Through incorporating information in T cell receptor clonal expansion and sharing, we have successfully established connections between distinct cell subtypes and shed light on their phenotypic and functional transitions. Notably, our approach characterizes three distinct exhausted T cell subtypes and reveals diverse transcriptome and clonal sharing patterns in autoimmune and immune-related adverse event inflammation. Furthermore, scAtlasVAE facilitates the automatic annotation of CD8+ T cell subtypes in query single-cell RNA sequencing datasets, enabling unbiased and scalable analyses. In conclusion, our work presents a comprehensive single-cell reference and computational framework for CD8+ T cell research.

Human CD8+ T cell map with single-cell transcriptome and TCR information.

Human CD8+ T cell map with single-cell transcriptome and TCR information.

Circulating tumor-reactive KIR+CD8+ T cells suppress anti-tumor immunity in patients with melanoma.

Effective anti-tumor immunity is driven by cytotoxic CD8+ T cells with specificity for tumor antigens. However, the factors that control successful tumor rejection are not well understood. Here we identify a subpopulation of CD8+ T cells that are tumor-antigen-specific and can be identified by KIR expression but paradoxically impair anti-tumor immunity in patients with melanoma. These tumor-antigen-specific KIR+CD8+ regulatory T cells target other tumor-antigen-specific CD8+ T cells, can be detected in both the tumor and the blood, have a conserved transcriptional program and are associated with a poor overall survival. These findings broaden our understanding of the transcriptional and functional heterogeneity of human CD8+ T cells and implicate KIR+CD8+ regulatory T cells as a cellular mediator of immune evasion in human cancer.

Polyfunctional T cells and unique cytokine clusters imprint the anti rAAV2/rAAV9 vector immune response.

Polyfunctional T cells programmed to perform activities such as degranulation of lytic enzymes and simultaneous production of multiple cytokines are associated with more effective control of viral infections. Immune responses to recombinant adeno-associated virus (rAAV) vector delivery systems can critically influence therapeutic efficacy and safety of gene therapy. However, knowledge of polyfunctional T cells in anti-AAV immune responses is scarce. To bridge this knowledge gap, we have investigated the polyfunctionality of primary human CD4 T cells from healthy donors after in-vitro exposure to rAAV2 or rAAV9 vectors. By performing proliferation assays of co-cultured T cells and rAAV pulsed monocyte-derived dendritic cells from healthy donors we demonstrate T cell reactivity of 43% and 50% to rAAV2 and rAAV9 vectors, respectively. We validated this frequency in a second screen using another set of healthy donors measuring CD25 and CD71 T cell activation. Single T cell secretome analysis of reactive donors uncovered a Th1 pro-inflammatory, cytolytic and chemoattractive cytokine release profile after stimulation with rAAV2 or rAAV9 vectors. 12.4% and 9.6% of the stimulated T cells displayed a polyfunctional cytokine response, respectively, including elevated polyfunctional inflammatory indices. These responses were characterized by cytokine clusters such as Granzyme B, MIP1-α and TNF-α released in combination by single T cells. Overall, our results provide insights into adaptive immunity with rAAV vector serotypes which will be important in advancing gene therapy safety, vector selection, immunogenicity assessment and better patient selection for AAV gene therapy.

Single-cell sequencing of full-length transcripts and T-cell receptors with automated high-throughput Smart-seq3

We developed an automated high-throughput Smart-seq3 (HT Smart-seq3) workflow that integrates best practices and an optimized protocol to enhance efficiency, scalability, and method reproducibility. This workflow consistently produces high-quality data with high cell capture efficiency and gene detection sensitivity. In a rigorous comparison with the 10X platform using human primary CD4 + T-cells, HT Smart-seq3 demonstrated higher cell capture efficiency, greater gene detection sensitivity, and lower dropout rates. Additionally, when sufficiently scaled, HT Smart-seq3 achieved a comparable resolution of cellular heterogeneity to 10X. Notably, through T-cell receptor (TCR) reconstruction, HT Smart-seq3 identified a greater number of productive alpha and beta chain pairs without the need for additional primer design to amplify full-length V(D)J segments, enabling more comprehensive TCR profiling across a broader range of species. Taken together, HT Smart-seq3 overcomes key technical challenges, offering distinct advantages that position it as a promising solution for the characterization of single-cell transcriptomes and immune repertoires, particularly well-suited for low-input, low-RNA content samples.

IL-12 drives the expression of the inhibitory receptor NKG2A on human tumor-reactive CD8 T cells

Blockade of NKG2A/HLA-E interaction is a promising strategy to unleash the anti-tumor response. Yet the role of NKG2A+ CD8 T cells in the anti-tumor response and the regulation of NKG2A expression on human tumor-infiltrating T cells are still poorly understood. Here, by performing CITE-seq on T cells derived from head and neck squamous cell carcinoma and colorectal cancer, we show that NKG2A expression is induced on CD8 T cells differentiating into cytotoxic, CD39+CD103+ double positive (DP) cells, a phenotype associated with tumor-reactive T cells. This developmental trajectory leads to TCR repertoire overlap between the NKG2A– and NKG2A+ DP CD8 T cells, suggesting shared antigen specificities. Mechanistically, IL-12 is essential for the expression of NKG2A on CD8 T cells in a CD40/CD40L- dependent manner, in conjunction with TCR stimulation. Our study thus reveals that NKG2A is induced by IL-12 on human tumor-reactive CD8 T cells exposed to a TGF-β-rich environment, highlighting an underappreciated immuno-regulatory feedback loop dependent on IL-12 stimulation.

Quantification of heterogeneity in human CD8+ T cell responses to vaccine antigens: an HLA-guided perspective.

Vaccines have historically played a pivotal role in controlling epidemics. Effective vaccines for viruses causing significant human disease, e.g., Ebola, Lassa fever, or Crimean Congo hemorrhagic fever virus, would be invaluable to public health strategies and counter-measure development missions. Here, we propose coverage metrics to quantify vaccine-induced CD8+ T cell-mediated immune protection, as well as metrics to characterize immuno-dominant epitopes, in light of human genetic heterogeneity and viral evolution. Proof-of-principle of our approach and methods are demonstrated for Ebola virus, SARS-CoV-2, and Burkholderia pseudomallei (vaccine) proteins.

Inducing and regulating human naive CD4+ T cell proliferation by different antigen presenting cells

We have shown in previous studies that naive CD4+ T cells isolated from human peripheral blood are induced to proliferate by CD4negCD11c+CD14negCD16neg dendritic cells presenting the superantigen SEE. Since this population is very poorly expressed in blood, we tried to find other antigen presenting cells (APCs) to induce this proliferation. The aim of the previous studies was to investigate the regulation of T cell proliferation in pediatric monogenic autoimmune diseases and the regulation of this proliferation by regulatory T cells (TREGs). Since the blood samples from pediatric patients were very small, it was important to study other APCs that are more commonly present in the blood. In this study we tested different APCs isolated from controls, CD19+ B cells, CD11c+CD14+ and CD11c+CD14neg monocytes, CD11c+CD14negCD16+ and CD16neg dendritic cells. The different T cell populations, naive effector T cells and regulatory T cells were separated simultaneously from the same sample. We show in these studies that CD19+ B cells, CD14neg and more specifically CD14negCD16+, are also able to induce T cell proliferation as previously described with CD14negCD16neg DCs, but under different conditions. No proliferation was induced with CD14+ monocytes. However, these three APCs are less potent than CD16neg and inhibition by TREG is more difficult to detect. In addition, when we test the role of CTLA-4 in the regulation of TEFF proliferation, we observe that for some APCs, the inhibition by CTLA-4 was quite different. No inhibition was observed with CD19+ B cells in contrast to CD11c+CD14negCD16+ and CD11c+CD14negCD16neg.

The apoptotic and anti-proliferative effects of Neosetophomone B in T-cell acute lymphoblastic leukaemia via PI3K/AKT/mTOR pathway inhibition.

The phosphatidylinositol 3-kinase/Protein Kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signalling pathway is pivotal in various cancers, including T-cell acute lymphoblastic leukaemia (T-ALL), a particularly aggressive type of leukaemia. This study investigates the effects of Neosetophomone B (NSP-B), a meroterpenoid fungal metabolite, on T-ALL cell lines, focusing on its anti-cancer mechanisms and therapeutic potential. NSP-B significantly inhibited the proliferation of T-ALL cells by inducing G0/G1 cell cycle arrest and promoting caspase-dependent apoptosis. Additionally, NSP-B led to the dephosphorylation and subsequent inactivation of the PI3K/AKT/mTOR signalling pathway, a critical pathway in cell survival and growth. Molecular docking studies revealed a strong binding affinity of NSP-B to the active site of AKT, primarily involving key residues crucial for its activity. Interestingly, NSP-B treatment also induced apoptosis and significantly reduced proliferation in phytohemagglutinin-activated primary human CD3+ T cells, accompanied by a G0/G1 cell cycle arrest. Importantly, NSP-B did not affect normal primary T cells, indicating a degree of selectivity in its action, targeting only T-ALL cells and activated T cells. In conclusion, our findings highlight the potential of NSP-B as a novel therapeutic agent for T-ALL, specifically targeting the aberrantly activated PI3K/AKT/mTOR pathway and being selective in action. These results provide a strong basis for further investigation into NSP-B's anti-cancer properties and potential application in T-ALL clinical therapies.

Abstract 4137500: The Histone Methyltransferase MLL1 Regulates Notch Signaling and T Cell Phenotype During Pathologic Abdominal Aortic Aneurysm Development

Objective: Abdominal aortic aneurysms (AAA) are characterized by pathological vascular remodeling and an imbalance between anti-inflammatory regulatory T H cells (Tregs) and pro-inflammatory T H 17 cells within the aortic wall. The mechanisms regulating CD4 + T H cell differentiation during AAA development remain unknown. Recently, the histone methyltransferase, mixed lineage leukemia 1 (MLL1), has been shown to influence T H cell phenotype by directly altering key transcription factors for T H 17 (RORγ) and Treg (FOXp3) differentiation in varying disease states. As such the objective of this study was to evaluate the role of MLL1 in promoting T H 17 activity within AAA development and to determine key T H 17 cell signaling pathways that are altered Methods: Single-cell sequencing was conducted on human AAAs and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and fed a saturated fat diet followed by either AngII infusion to induce AAAs (1 µg/kg/min) or saline. Mice with T-cell specific deletion of Notch1 signaling (Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) were subjected to the AngII-induced AAA model and AAA diameters quantified. CD4+ T-cells were isolated by MACs and gene expression analyzed by qPCR as well as T-cell phenotype by flow cytometry. ChIP was used to evaluate histone 3 lysine trimethylation (H3K4me3). Results: Single-cell RNA sequencing of human aortic tissue revealed that the epigenetic enzyme MLL1 and Notch1 signaling were profoundly upregulated in human AAA CD4+ cells compared to non-aneurysmal control samples. Congruently, CD4 + T H cells isolated from the murine AngII-induced AAA model displayed a predominance of T H 17 phenotype and elevated expression of MLL1. Histone methylation was evaluated by ChIP in AngII-induced AAA CD4+ cells and demonstrated increased levels of the MLL1-mediated activation histone methylation mark, H3K4me3, on the Notch 1 promoter. Mice deficient in CD4 + T H cell Notch signaling ( Notch1 f/f CD4 cre+ ) or MLL1 ( Mll1 f/f CD4 cre+ ) subjected to the AngII-induced murine model displayed significant reduction AAA development (n=10/group, p<0.05) with decreased CD4+ T H 17 cell activity and reciprocal increase in Treg expression. Conclusions: MLL1, a histone methyltransferase, regulates Notch1 signaling and T-cell differentiation in AAA development, causing pathologic inflammation. This pathway may serve as a potential therapeutic target to prevent aortic dilation.

TMIC-50. IMPACT OF ZOTIRACICLIB ON GLIOMA TUMOR MICROENVIRONMENT

Abstract BACKGROUND Zotiraciclib, an orally administered multi-kinase inhibitor, has shown anti-glioma effect via suppression of transcriptional process and mitochondrial function. Here, we investigate the impact of zotiraciclib on the glioma immune microenvironment. METHODS Mouse glioma was generated by intracranial injection of mouse syngeneic cells (IDH1-wildtype NPA cells or IDH1R132H-mutant NPAI cells) in C57BL/6 mice. Zotiraciclib was administered (30 mg/kg, intraperitoneal injection, twice per week) starting seven days after tumor implantation. NPA and NPAI tumors were dissected after 10 and 22 days’ treatment, respectively. Immune cells in the tumor tissue were isolated and examined using flow cytometry. Primary human CD8+ cells and CD14+ monocytes isolated from the blood of a healthy donor were used to further investigate the effect of zotiraciclib (15 nM and 50 nM) on human cytotoxic T cell activation and immunosuppressive M2 macrophage polarization, respectively, using flow cytometry. RESULTS No significant difference in the abundance of T cells, myeloid cells, NK cells, NKT cells, DCs, and B cells between zotiraciclib-treated and vehicle-treated groups was detected in neither NPA nor NPAI tumors. The treatment-induced change in the abundance of cytotoxic T cells, helper T cells, macrophages, monocytes, neutrophils, M-MDSCs, PMN-MDSCs, Tregs, and M2 macrophages wasn’t observed. Zotiraciclib reduced the expansion of human CD8+ cytotoxic T cells, but did not affect the expression of CD25, HLA-DR, TNFα and IFNγ after activation in vitro. The viability of human M2 macrophages during differentiation and polarization was not affected, while the expression of CD163 and CD206 was reduced by zotiraciclib. CONCLUSIONS The immunophenotyping performed for this study demonstrates that zotiraciclib does not alter the mouse glioma immune microenvironment. Complementary studies using human immune cells shows that zotiraciclib does not suppress the activation of cytotoxic T cells but reduces the polarization of immunosuppressive M2 macrophages, supporting combining zotiraciclib and immune stimulatory approach in glioma treatment.

TMOD-31. SINGLE-CELL PROFILING AND CHARACTERIZATION OF PATIENT-DERIVED XENOGRAFT GLIOBLASTOMA MODEL IN HUMANIZED MICE

Abstract Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in adults, and the current standard therapy has limited efficacy. To develop effective therapies, it is desirable to develop an animal model that can replicate the tumor heterogeneity and immune microenvironment of human GBM. Here, we describe a novel GBM mouse model established with a patient-derived xenograft (PDX) in humanized mice harboring a nearly complete human immune environment. Humanized mice were generated by engrafting human CD34+ hematopoietic stem cells from umbilical cord blood into immunocompromised mice (NSG, NSG-SGM3, and NOG-EXL) after myeloablation. NSG-SGM3 is a triple transgenic mouse encoding human IL3, GM-CSF, and SCF on the NSG strain background. NOG-EXL is a NOG strain with human IL3 and GM-CSF expression. These cytokines foster the development of the human myeloid lineage and its differentiation. NSG-SGM3 and NOG-EXL were superior in immune cell reproducibility, especially myeloid cells. Radioresistant PDX GBM cells established after repeated irradiation were injected into the forebrain of humanized mice and the same immunocompromised mice without humanization. Human PDX GBM cells formed aggressive tumors with similar growth speed in the humanized mice compared to non-humanized mice; however, their dissemination to the spinal cord was significantly suppressed in humanized mice, suggesting that human immune cells impact the spinal dissemination of GBM cells. Single-cell RNA sequencing analysis revealed infiltration of human CD4+ T, CD8+ T, NK cells, and macrophages, as well as infiltration of regulatory T and exhausted CD8+ cells into the brain tumor, reflecting the immunosuppressive landscape of recurrent human GBM. Furthermore, we found that tumor heterogeneity was increased in humanized mice compared to non-humanized mice. There was a notable rise in neural progenitor-like cell populations, indicating tumor adaptation to immune pressures. This novel GBM-PDX humanized mouse model might be highly beneficial for analyzing the interaction between human tumor cells and human immune cells, and evaluating the effect of immunotherapies.

IFN-γ derived from activated human CD4+ T cells inhibits the replication of SARS-CoV-2 depending on cell-type and viral strain

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and vaccination elicit both T cell and B cell immune responses in immunocompetent individuals. However, the mechanisms underlying the antiviral effects mediated by CD4+ T cells are not fully understood. In this study, we analyzed the culture supernatant (SN) from polyclonally stimulated human CD4+ T cells as a model for soluble mediators derived from SARS-CoV-2-stimulated CD4+ T cells. Interestingly, this SN inhibited SARS-CoV-2 propagation in a viral strain- and host cell type-dependent manner. The original wild-type showed the highest susceptibility, whereas the Delta variant exhibited resistance in the human monocyte cell line. In addition, antibody-dependent enhancement (ADE) of infection with the original strain was also abolished in the presence of the SN. The findings showed that the inhibitory effect on viral propagation by the SN was mostly attributed to interferon-γ (IFN-γ) that was present in the SN. These results highlight the potential role of IFN-γ as an anti-SARS-CoV-2 mediator derived from CD4+ T cells, and suggest that we need to understand the SARS-CoV-2 strain-dependent sensitivity to IFN-γ in controlling clinical outcomes. In addition, characterization of new SARS-CoV-2 variants in terms of IFN-γ-sensitivity will have important implications for selecting therapeutic strategies.

Dental pulp mesenchymal stem cell (DPSCs)-derived soluble factors, produced under hypoxic conditions, support angiogenesis via endothelial cell activation and generation of M2-like macrophages

BackgroundCell therapy has emerged as a revolutionary tool to repair damaged tissues by restoration of an adequate vasculature. Dental Pulp stem cells (DPSC), due to their easy biological access, ex vivo properties, and ability to support angiogenesis have been largely explored in regenerative medicine.MethodsHere, we tested the capability of Dental Pulp Stem Cell-Conditioned medium (DPSC-CM), produced in normoxic (DPSC-CM Normox) or hypoxic (DPSC-CM Hypox) conditions, to support angiogenesis via their soluble factors. CMs were characterized by a secretome protein array, then used for in vivo and in vitro experiments. In in vivo experiments, DPSC-CMs were associated to an Ultimatrix sponge and injected in nude mice. After excision, Ultimatrix were assayed by immunohistochemistry, electron microscopy and flow cytometry, to evaluate the presence of endothelial, stromal, and immune cells.For in vitro procedures, DPSC-CMs were used on human umbilical-vein endothelial cells (HUVECs), to test their effects on cell adhesion, migration, tube formation, and on their capability to recruit human CD14+ monocytes.ResultsWe found that DPSC-CM Hypox exert stronger pro-angiogenic activities, compared with DPSC-CM Normox, by increasing the frequency of CD31+ endothelial cells, the number of vessels and hemoglobin content in the Ultimatrix sponges. We observed that Utimatrix sponges associated with DPSC-CM Hypox or DPSC-CM Normox shared similar capability to recruit CD45− stromal cells, CD45+ leukocytes, F4/80+ macrophages, CD80+ M1-macrophages and CD206+ M2-macropages. We also observed that DPSC-CM Hypox and DPSC-CM Normox have similar capabilities to support HUVEC adhesion, migration, induction of a pro-angiogenic gene signature and the generation of capillary-like structures, together with the ability to recruit human CD14+ monocytes.ConclusionsOur results provide evidence that DPSCs-CM, produced under hypoxic conditions, can be proposed as a tool able to support angiogenesis via macrophage polarization, suggesting its use to overcome the issues and restrictions associated with the use of staminal cells.

A temperature-sensitive and less immunogenic Sendai virus for efficient gene editing.

Gene editing has the potential to be a powerful tool for the treatment of human diseases including HIV, β-thalassemias, and sickle cell disease. Recent advances have begun to overcome one of the major limiting factors of this technology, namely delivery of the CRISPR-Cas9 gene editing machinery, by utilizing viral vectors. However, gene editing therapies have yet to be implemented due to inherent risks associated with the DNA viral vectors typically used for delivery. As an alternative strategy, we have developed an RNA-based Sendai virus CRISPR-Cas9 delivery vector that does not integrate into the genome, is temperature sensitive, and does not induce a significant host interferon response. This recombinant SeV successfully delivered CRISPR-Cas9 in primary human CD14+ monocytes ex vivo resulting in a high level of CCR5 editing and inhibition of HIV infection.