Search

Drug-based antiretroviral therapies (ART) efficiently suppress HIV replication in humans, but the virus persists as integrated proviral reservoirs in small numbers of cells. Importantly, ART cannot eliminate HIV from an infected individual, since it does not target the integrated provirus. Therefore, genome editing-based strategies that can inactivate or excise HIV genomes would provide the technology for novel curative therapies. In fact, the HIV-1 LTR-specific designer-recombinase Brec1 has been shown to remove integrated proviruses from infected cells and is highly efficacious on clinical HIV-1 isolates in vitro and in vivo, suggesting that Brec1 has the potential for clinical development of advanced HIV-1 eradication strategies in people living with HIV. In line with the preparation of a first-in-human advanced therapy medicinal product gene therapy trial, we here present an extensive preclinical evaluation of Brec1 and lentiviral vectors expressing the Brec1 transgene. This included detailed functional analysis of potential genomic off-target sites, assessing vector safety by investigating vector copy number (VCN) and the risk for potential vector-related insertional mutagenesis, as well as analyzing the potential of Brec1 to trigger an undesired strong T cell immune response. In conclusion, the antiviral designer-recombinase Brec1 is shown to lack any detectable cytopathic, genotoxic or T cell-related immunogenic effects, thereby meeting an important precondition for clinical application of the therapeutic lentiviral vector LV-Brec1 in novel HIV-1 curative strategies., Competing Interests: The author’s have read the journal’s policy and have the following competing interests: Jan Chemnitz, Joachim Hauber, Frank Buchholz, Ilona Hauber, Niklas Beschorner and Maike Voges are founding partners of PROVIREX Genome Editing Therapies GmbH, which is commercially developing Brec1 technology. Niklas Beschorner and Maike Voges are full-time employees of PROVIREX Genome Editing Therapies GmbH since 2022, and Jan Chemnitz, Frank Buchholz, Joachim Hauber and Ilona Hauber are part-time employees. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare., (Copyright: © 2024 Beschorner et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

CD8 + T cells are a crucial part of the adaptive immune system, responsible for combating intracellular pathogens and tumor cells. The initial activation of T cells involves the formation of highly dynamic Ca 2+ microdomains. Recently, purinergic signaling was shown to be involved in the formation of the initial Ca 2+ microdomains in CD4 + T cells. In this study, the role of purinergic cation channels, particularly P2X4 and P2X7, in CD8 + T cell signaling from initial events to downstream responses was investigated, focusing on various aspects of T cell activation, including Ca 2+ microdomains, global Ca 2+ responses, NFAT-1 translocation, cytokine expression, and proliferation. While Ca 2+ microdomain formation was significantly reduced in the first milliseconds to seconds in CD8 + T cells lacking P2X4 and P2X7 channels, global Ca 2+ responses over minutes were comparable between wild-type (WT) and knockout cells. However, the onset velocity was reduced in P2X4-deficient cells, and P2X4, as well as P2X7-deficient cells, exhibited a delayed response to reach a certain level of free cytosolic Ca 2+ concentration ([Ca 2+ ] i ). NFAT-1 translocation, a crucial transcription factor in T cell activation, was also impaired in CD8 + T cells lacking P2X4 and P2X7. In addition, the expression of IFN-γ, a major pro-inflammatory cytokine produced by activated CD8 + T cells, and Nur77, a negative regulator of T cell activation, was significantly reduced 18h post-stimulation in the knockout cells. In line, the proliferation of T cells after 3 days was also impaired in the absence of P2X4 and P2X7 channels. In summary, the study demonstrates that purinergic signaling through P2X4 and P2X7 enhances initial Ca 2+ events during CD8 + T cell activation and plays a crucial role in regulating downstream responses, including NFAT-1 translocation, cytokine expression, and proliferation on multiple timescales. These findings suggest that targeting purinergic signaling pathways may offer potential therapeutic interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Brock, Lory, Möckl, Birus, Stähler, Woelk, Jaeckstein, Heeren, Koch-Nolte, Rissiek, Mittrücker, Guse, Werner and Diercks.)

The transcription factor Interferon Regulatory Factor 4 (IRF4) is central in control of T cell activation and differentiation. Deficiency of IRF4 results in severe immune deficiency and affects maturation and function of most if not all T cell subsets. Here we use mouse infection models for Citrobacter rodentium and Strongyloides ratti to analyze the function of IRF4 in T helper (Th) 17 and Th2 cell responses, respectively. IRF4 deficient mice were impaired in the control of both pathogens, failed to mount Th17 and Th2 cell responses and showed impaired recruitment of T helper cells to the intestine, the infection site of both pathogens. Compromised intestinal migration was associated with reduced expression of the intestinal homing receptors α4β7 integrin, CCR9 and GPR15. Identification of IRF4 binding sites in the gene loci of these receptors suggests a direct control of their expression by IRF4. Competitive T cell transfer assays further demonstrated that loss of one functional Irf4 allele already affected intestinal accumulation and Th2 and Th17 cell generation, indicating that lower IRF4 levels are of disadvantage for Th2 and Th17 cell differentiation as well as their migration to the intestine. Conversion of peripheral CD4 + T cells from an Irf4 wildtype to an Irf4 heterozygous or from an Irf4 heterozygous to a homozygous mutant genotype after C. rodentium or S. ratti infection did not reduce their capacity to produce Th17 or Th2 cytokines and only partially affected their persistence in the intestine, revealing that IRF4 is not essential for maintenance of the Th2 and Th17 phenotype and for survival of these T helper cells in the intestine. In conclusion, we demonstrate that the expression levels of IRF4 determine Th2 and Th17 cell differentiation and their intestinal accumulation but that IRF4 expression is not crucial for Th2 and Th17 cell survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schmidt, Harberts, Reimers, Bertram, Voß, Schmid, Lory, Spohn, Koch-Nolte, Huber, Raczkowski, Breloer and Mittrücker.)

γδ T cells are involved in the control of Staphylococcus aureus infection, but their importance in protection compared to other T cells is unclear. We used a mouse model of systemic S. aureus infection associated with high bacterial load and persistence in the kidney. Infection caused fulminant accumulation of γδ T cells in the kidney. Renal γδ T cells acquired tissue residency and were maintained in high numbers during chronic infection. At day 7, up to 50% of renal γδ T cells produced IL-17A in situ and a large fraction of renal γδ T cells remained IL-17A + during chronic infection. Controlled depletion revealed that γδ T cells restricted renal S. aureus replication in the acute infection and provided protection during chronic renal infection and upon reinfection. Our results demonstrate that kidney-resident γδ T cells are nonredundant in limiting local S. aureus growth during chronic infection and provide enhanced protection against reinfection.

In the past, proinflammatory CD11b + Ly6C hi monocytes were predominantly considered as a uniform population. However, recent investigations suggests that this population is far more diverse than previously thought. For example, in mouse models of Entamoeba (E.) histolytica and Listeria (L.) monocytogenes liver infections, it was shown that their absence had opposite effects. In the former model, it ameliorated parasite-dependent liver injury, whereas in the listeria model it exacerbated liver pathology. Here, we analyzed Ly6C hi monocytes from the liver of both infection models at transcriptome, protein, and functional levels. Paralleled by E. histolytica - and L. monocytogenes -specific differences in recruitment-relevant chemokines, both infections induced accumulation of Ly6C + monocytes at infection sites. Transcriptomic analysis revealed a high similarity between monocytes from naïve and parasite-infected mice and a clear proinflammatory phenotype of listeria-induced monocytes. This was further reflected by the upregulation of M2-related transcription factors (e.g., Mafb, Nr4a1, Fos ) and higher CD14 expression by Ly6C hi monocytes in the E. histolytica infection model. In contrast, monocytes from the listeria infection model expressed M1-related transcription factors (e.g., Irf2, Mndal, Ifi204 ) and showed higher expression of CD38, CD74, and CD86, as well as higher ROS production. Taken together, proinflammatory Ly6C hi monocytes vary considerably depending on the causative pathogen. By using markers identified in the study, Ly6C hi monocytes can be further subdivided into different populations.

Antigen recognition by the T-cell receptor induces a cytosolic Ca 2+ signal that is crucial for T-cell function. The Ca 2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca 2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca 2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro , Trpm2 -/- and WT CD4 + and CD8 + T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2 -/- CD8 + T cells and unimpaired differentiation of CD4 + T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo , Trpm2 -/- and WT CD8 + T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2 -/- mice and after transfer of WT and Trpm2 -/- CD8 + T cells into infected recipients. CD4 + T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2 -/- CD4 + T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lory, Nawrocki, Corazza, Schmid, Schumacher, Bedke, Menzel, Koch-Nolte, Guse, Huber and Mittrücker.)