James H. Felce

Background: The leukaemia-derived Jurkat E6.1 cell line has been used as a model T cell in the study of many aspects of T cell biology, most notably activation in response to T cell receptor (TCR) engagement. Methods: We present whole-transcriptome RNA-Sequencing data for Jurkat E6.1 cells in the resting state and two hours post-activation via TCR and CD28. We compare early transcriptional responses in the presence and absence of the chemokines CXCL12 and CCL19, and perform a basic comparison between observed transcriptional responses in Jurkat E6.1 cells and those in primary human T cells using publicly deposited data. Results: Jurkat E6.1 cells have many of the hallmarks of standard T cell transcriptional responses to activation, but lack most of the depth of responses in primary cells. Conclusions: These data indicate that Jurkat E6.1 cells hence represent only a highly simplified model of early T cell transcriptional responses.

The dual anticytokine and antiviral actions of baricitinib reduce SARS-CoV-2 infectivity in organoids and morbidity in people.

The Plasmodium species that cause malaria are obligate intracellular parasites, and disease symptoms occur when these parasites replicate in human blood. Despite the risk of immune detection, the parasite delivers proteins that bind to host receptors on the cell surfaces of infected erythrocytes. In the causative parasite of the most deadly form of malaria in humans, Plasmodium falciparum, RIFINs form the largest family of surface proteins displayed by erythrocytes¹. Some RIFINs can bind to inhibitory immune receptors, and these RIFINs act as targets for unusual antibodies that contain a LAIR1 ectodomain^2,3,4 or as ligands for LILRB1⁵. RIFINs stimulate the activation of and signalling by LILRB1⁵, which could potentially lead to the dampening of human immune responses. Here, to understand how RIFINs activate LILRB1-mediated signalling, we determine the structure of a RIFIN bound to LILRB1. We show that this RIFIN mimics the natural activating ligand of LILRB1, MHC class I, in its LILRB1-binding mode. A single mutation in the RIFIN disrupts the complex, blocks LILRB1 binding of all tested RIFINs and abolishes signalling in a reporter assay. In a supported lipid bilayer system, which mimics the activation of natural killer (NK) cells by antibody-dependent cell-mediated cytotoxicity, both RIFIN and MHC are recruited to the immunological synapse of NK cells and reduce the activation of NK cells, as measured by the mobilization of perforin. Therefore, LILRB1-binding RIFINs mimic the binding mode of the natural ligand of LILRB1 and suppress the function of NK cells.

Background: The leukaemia-derived Jurkat E6.1 cell line has been used as a model T cell in the study of many aspects of T cell biology, most notably activation in response to T cell receptor (TCR) engagement. Methods: We present whole-transcriptome RNA-Sequencing data for Jurkat E6.1 cells in the resting state and two hours post-activation via TCR and CD28. We compare early transcriptional responses in the presence and absence of the chemokines CXCL12 and CCL19, and perform a basic comparison between observed transcriptional responses in Jurkat E6.1 cells and those in primary human T cells using publicly deposited data. Results: Jurkat E6.1 cells have many of the hallmarks of standard T cell transcriptional responses to activation, but lack most of the depth of responses in primary cells. Conclusions: These data indicate that Jurkat E6.1 cells hence represent only a highly simplified model of early T cell transcriptional responses.

We examined the potential value of the natural killer (NK) cell line; NK-92, as immunotherapy tool for breast cancer (BC) treatment and searched for biomarker(s) of sensitivity to NK-92-mediated cytotoxicity. The cytotoxic activity of NK-92 cells towards one breast precancerous and nine BC cell lines was analyzed using calcein-AM and degranulation assays. The molecules associated with NK-92-responsiveness were determined by differential gene expression analysis using RNA-sequencing and validated by RT-PCR, immunostaining and flow cytometry. NK-target interactions and immunological synapse formation were assessed by fluorescence microscopy. Potential biomarker expression was determined by IHC in 99 patient-derived BC tissues and 10 normal mammary epithelial tissues. Most (8/9) BC cell lines were resistant while only one BC and the precancerous cell lines were effectively killed by NK-92 lymphocytes. NK-92-sensitive target cells specifically expressed CD56, which ectopic expression in CD56-negative BC cells induced their sensitivity to NK-92-mediated killing, suggesting that CD56 is not only a biomarker of responsiveness but actively regulates NK function. CD56 adhesion molecules which are also expressed on NK cells accumulate at the immunological synapse enhancing NK-target interactions, cytotoxic granzyme B transfer from NK-92 to CD56-expressing target cells and induction of caspase 3 activation in targets. Interestingly, CD56 expression was found to be reduced in breast tumor tissues (36%) with strong inter- and intratumoral heterogeneity in comparison to normal breast tissues (80%). CD56 is a potential predictive biomarker for BC responsiveness to NK-92-cell based immunotherapy and loss of CD56 expression might be a mechanism of escape from NK-immunity.

How G protein-coupled receptors are assembled is a matter of considerable interest owing in large part to their remarkable pharmacological importance. For determining receptor stoichiometry, resonance energy transfer-based methods offer considerable advantages insofar as they provide the necessary spatial resolution, and because measurements can be made in situ, relatively easily. This chapter describes three complementary stoichiometric assays that rely on measurements of bioluminescence resonance energy transfer. These quantitative approaches make it possible to identify true protein–protein interactions from non-specific associations that inevitably result from constraining proteins in cellular membranes. In our experience, concordant data obtained in two or more of these assays, benchmarked with suitable controls, strongly predict receptor stoichiometry.

Exclusion of a phosphatase from regions of antigen-receptor engagement facilitates mast cell activation.

Natural killer cells target antibody-bound cells following engagement of the Fc receptor CD16. Srpan et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201712085) reveal that activation-induced shedding of CD16 leads to more motile behavior, allowing more targets to be engaged and killed in a given time.

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. It is important, therefore, to be able to elucidate molecular processes occurring at these interfaces. However, the detailed investigation of each component’s contribution to the formation and regulation of the contact is hampered by the complexity of cellular composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially for using advanced imaging technology. One approach to circumventing these problems is to establish in vitro systems that faithfully mimic immune cell interactions, incorporating complexity that can be ‘dialled-in’ as needed. Here, we present an in vitro system making use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we begin to investigate the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and the intracellular rearrangements that accompany the initiation of signalling in T cells. The model system presented here is expected to be widely applicable. Summary Statement: Immune cell-cell interactions are reconstituted in free-standing vesicles wherein spatiotemporal aspects of immune synapse formation can be investigated.