LAIR1 prevents excess inflammatory tissue damage in S. aureus skin infection and Cutaneous T-cell Lymphoma.

Patients with cutaneous T cell lymphoma (CTCL) experience high morbidity and mortality due to S. aureus skin infections and sepsis, but the causative immune defect is unclear. We previously identified high levels of LAIR2, a decoy protein for the inhibitory receptor LAIR1, in advanced CTCL. Mice do not have a LAIR2 homolog, so we used Lair1 knock-out (KO) mice to model LAIR2 overexpression. In a model of subcutaneous S. aureus skin infection, Lair1 KO mice had significantly larger abscesses and areas of dermonecrosis compared to WT. Lair1 KO exhibited a pattern of increased inflammatory responses in infection and sterile immune stimulation, including increased production of proinflammatory cytokines and myeloid chemokines, neutrophil ROS, and collagen/ECM remodeling pathways. Notably, Lair1 KO infected skin had a similar bacterial burden and neutrophils and monocytes had equivalent S. aureus phagocytosis compared to WT. These findings support a model in which lack of LAIR1 signaling causes an excessive inflammatory response that does not improve infection control. CTCL skin lesions harbored similar patterns of increased expression in cytokine and collagen/ECM remodeling pathways, suggesting that high levels of LAIR2 in CTCL recapitulates Lair1 KO, causing inflammatory tissue damage and compromising host defense against S. aureus infection.

Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation.

The surface receptor CD8α is present on 20-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses for leukemia patients in prior studies, thus we hypothesized that CD8α may impact critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models, compared to CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These 'induced' CD8α+ ('iCD8α+') NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity, compared to those that sustained existing CD8α expression ('sustained CD8α+) or those that remained CD8α- ('persistent CD8α-'). These iCD8α+ cells originated from an IL-15Rß high NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identifies human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion and exhibits a suppressive effect on NK cell activating receptors.

LPS and type I and II interferons have opposing effects on epigenetic regulation of LAIR1 expression in mouse and human macrophages.

Inhibitory immune receptors are important for maintaining immune homeostasis. We identified epigenetic alterations in 2 members of this group, LAIR1 and LAIR2, in lymphoma patients with inflammatory tissue damage and susceptibility to infection. We predicted that the expression of LAIR genes is controlled by immune mediators acting on transcriptional regulatory elements. Using flow cytometry, quantitative reverse-transcription polymerase chain reaction, and RNA sequencing, we measured LAIR1 and LAIR2 in human and murine immune cell subsets at baseline and posttreatment with immune mediators, including type I and II interferons, tumor necrosis factor α, and lipopolysaccharide (LPS). We identified candidate regulatory elements using epigenome profiling and measured their regulatory activity using luciferase reporters. LAIR1 expression substantially increases during monocyte differentiation to macrophages in both species. In contrast, murine and human macrophages exhibited opposite changes in LAIR1 in response to immune stimuli: human LAIR1 increased with LPS while mouse LAIR1 increased with interferon γ. LAIR genes had distinct patterns of enhancer activity with variable responses to immune stimuli. To identify relevant transcription factors (TFs), we developed integrative bioinformatic techniques applied to TF chromatin immunoprecipitation sequencing, RNA sequencing, and luciferase activity, revealing distinct sets of TFs for each LAIR gene. Most strikingly, LAIR1 TFs include nuclear factor kappa B factors RELA and RELB, while Lair1 and LAIR2 instead include STAT3 and/or STAT5. Regulation by nuclear factor kappa B factors may therefore explain the LPS-induced increase in LAIR1 expression, in contrast to Lair1 decrease. Our findings reveal new insights into transcriptional mechanisms that control distinct expression patterns of LAIR genes in response to inflammatory stimuli in human and murine myeloid and lymphoid cells.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

Human respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

BCALM (AC099524.1) Is a Human B Lymphocyte-Specific Long Noncoding RNA That Modulates B Cell Receptor-Mediated Calcium Signaling.

Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 (PLCG2), a B cell-specific enzyme that stimulates intracellular Ca2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca+ signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca+ flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca+ signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca+ signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers.