Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions

Flow-FISH as a Tool for Studying Bacteria, Fungi and Viruses

Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs (fluorescently labeled) probes directed against DNA or (m)RNA, for instance targeting a gene or microorganism of interest and provides information on a single-cell level. Furthermore, by combining Flow-FISH with antibody-based protein detection, proteins of interest can be measured simultaneously with genetic material. Additionally, depending on the type of Flow-FISH assay, Flow-FISH can also be multiplexed, allowing for the simultaneous measurement of multiple gene targets and/or microorganisms. Together, this allows for, e.g., single-cell gene expression analysis or identification of (sub)strains in mixed cultures. Flow-FISH has been used in mammalian cells but has also been extensively employed to study diverse microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Specifically, the detection of (intracellular) pathogens, studying microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, with a particular focus on the viruses EBV, HIV-1, and SARS-CoV-2

Measuring T cell responses by flow cytometry–based fluorescence in situ hybridization

T cells produce a wide variety of effector molecules in response to infections, such as cytokines, chemokines, granzymes, and perforins. Because different stimuli promote the production of specific effector molecules, T cell responses come in different flavors. In addition, single-cell analysis of protein production revealed that T cells respond heterogeneously to activation. To unravel the regulatory mechanisms that determine T cell effector function, novel methods were developed that simultaneously measure protein levels with the corresponding mRNA. These flow cytometry-based fluorescence in situ hybridization (Flow-FISH) technologies allow for multiparameter analysis with single-cell resolution of both nucleic acids and proteins. Here, we review the currently available methods of Flow-FISH and describe the possible applications thereof, with a specific focus on T cells

Human T cells employ conserved AU-rich elements to fine-tune IFN-γ production

Long-lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate–rich elements (AREs) that are located in the 3′ untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3′ UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes

Critical role of post-transcriptional regulation for IFN-γ in tumor-infiltrating T cells

Protective T cell responses against tumors require the production of Interferon gamma (IFN-γ). However, tumor-infiltrating T cells (TILs) gradually lose their capacity to produce IFN-γ and therefore fail to clear malignant cells. Dissecting the underlying mechanisms that block cytokine production is thus key for improving T cell products. Here we show that although TILs express substantial levels of Ifng mRNA, post-transcriptional mechanisms impede the production of IFN-γ protein due to loss of mRNA stability. CD28 triggering, but not PD1 blocking antibodies, effectively restores the stability of Ifng mRNA. Intriguingly, TILs devoid of AU-rich elements within the 3ʹuntranslated region maintain stabilized Ifng mRNA and produce more IFN-γ protein than wild-type TILs. This sustained IFN-γ production translates into effective suppression of tumor outgrowth, which is almost exclusively mediated by direct effects on the tumor cells. We therefore conclude that post-transcriptional mechanisms could be modulated to potentiate effective T cell therapies in cancer

Costimulation through TLR2 Drives Polyfunctional CD8+ T Cell Responses

Optimal T cell activation requires Ag recognition through the TCR, engagement of costimulatory molecules, and cytokines. T cells can also directly recognize danger signals through the expression of TLRs. Whether TLR ligands have the capacity to provide costimulatory signals and enhance Ag-driven T cell activation is not well understood. In this study, we show that TLR2 and TLR7 ligands potently lower the Ag threshold for cytokine production in T cells. To investigate how TLR triggering supports cytokine production, we adapted the protocol for flow cytometry-based fluorescence in situ hybridization to mouse T cells. The simultaneous detection of cytokine mRNA and protein with single-cell resolution revealed that TLR triggering primarily drives de novo mRNA transcription. Ifng mRNA stabilization only occurs when the TCR is engaged. TLR2-, but not TLR7-mediated costimulation, can enhance mRNA stability at low Ag levels. Importantly, TLR2 costimulation increases the percentage of polyfunctional T cells, a hallmark of potent T cell responses. In conclusion, TLR-mediated costimulation effectively potentiates T cell effector function to suboptimal Ag levels

Critical role of post-transcriptional regulation for IFN-γ in tumor-infiltrating T cells

Protective T cell responses against tumors require the production of Interferon gamma (IFN-γ). However, tumor-infiltrating T cells (TILs) gradually lose their capacity to produce IFN-γ and therefore fail to clear malignant cells. Dissecting the underlying mechanisms that block cytokine production is thus key for improving T cell products. Here we show that although TILs express substantial levels of Ifng mRNA, post-transcriptional mechanisms impede the production of IFN-γ protein due to loss of mRNA stability. CD28 triggering, but not PD1 blocking antibodies, effectively restores the stability of Ifng mRNA. Intriguingly, TILs devoid of AU-rich elements within the 3ʹuntranslated region maintain stabilized Ifng mRNA and produce more IFN-γ protein than wild-type TILs. This sustained IFN-γ production translates into effective suppression of tumor outgrowth, which is almost exclusively mediated by direct effects on the tumor cells. We therefore conclude that post-transcriptional mechanisms could be modulated to potentiate effective T cell therapies in cancer

Memory CD8+ T cells upregulate glycolysis and effector functions under limiting oxygen conditions

Memory CD8+ T cells are indispensable for maintaining long-term immunity against intracellular pathogens and tumors. Despite their presence at oxygen-deprived infected tissue sites or in tumors, the impact of local oxygen pressure on memory CD8+ T cells remains largely unclear. We sought to elucidate how oxygen pressure impacts memory CD8+ T cells arising after infection with Listeria monocytogenes-OVA. Our data revealed that reduced oxygen pressure during in vitro culture switched CD8+ T cell metabolism from oxidative phosphorylation to a glycolytic phenotype. Quantitative proteomic analysis showed that limiting oxygen conditions increased the expression of glucose transporters and components of the glycolytic pathway, while decreasing TCA cycle and mitochondrial respiratory chain proteins. The altered CD8+ T cell metabolism did not affect the expansion potential, but enhanced the granzyme B and IFN-γ production capacity. In vivo, memory CD8+ T cells cultured under low oxygen pressure provided protection against bacterial rechallenge. Taken together, our study indicates that strategies of cellular immune therapy may benefit from reducing oxygen during culture to develop memory CD8+ T cells with superior effector functions