In this Top Read, Matsuyama et al. (p. 1532) reported that Streptococcus pneumoniae can drive excessive mucus formation in the middle ear in a TLR-independent manner, causing otitis media (OM). In both mice and cultured human middle ear epithelial cells, infection by several OM-causing S. pneumoniae strains that produce pneumolysin (PLY) induced phosphorylation of the kinase AMPKα1, whereas a PLY-deficient strain did not. PLY-dependent activation of AMPKα1 required CaMKKβ and MLK3 kinases, accessibility of the cholesterol-binding domains of PLY, and a cell membrane rich in cholesterol, but did not require cytoskeletal remodeling. AMPKα1 activation drove inhibition of JNK, which promotes PLY-dependent, S. pneumoniae–induced MUC5AC expression. Interestingly, PLY also activated AMPKα1 in cells that lacked functional TLR2/4-MyD88 signaling. Altogether, the study demonstrates the plausibility of developing a nonantibiotic therapeutic for S. pneumoniae–driven OM by targeting the negative cross-talk between TLR-independent AMPKα1 inhibition of JNK and JNK/TLR-dependent pathways.

In this Top Read, Agnihotri et al. (p. 1586) mapped the epitopes of select Abs known to block binding of hLAG3 to MHC class II, which are potential cancer immunotherapeutics. Biolayer interferometry revealed that the Fab or single-chain variable fragment of these seven mAbs bound to hLAG3 with nanomolar affinity. Competitive binding experiments, as well as epitope mapping assays using human and mouse LAG3 chimeras, showed that the mAbs bound four distinct epitopes within the extracellular Ig-like D1 domain of hLAG3, and that two noncompeting mAbs bound LAG3 simultaneously. Altogether, the data suggest that D1-targeting therapeutic mAbs block binding of hLAG3 to MHC class II by at least partially overlapping with the MHC class II binding site, and that using multiple mAbs with distinct D1 epitopes may provide improved anti–hLAG3-based treatment aimed at reawakening regulatory T cell function in cancer.

In this Top Read, Gao et al. (p. 1513) investigated the characteristics of naive and memory B cells in human patients with systemic lupus erythematosus (SLE) using single-cell RNA sequencing. In SLE patients, naive B cells become activated and are the precursors of pathogenic, atypical memory B cells. The authors showed that these naive B cells expressed type I IFN–associated genes, whereas healthy naive B cells expressed IL-4–associated genes. SLE B cells also showed diminished IL-4R expression, which correlated with autoantibody production. Furthermore, healthy activated naive B cells showed characteristics of germinal center and activated memory B cells. In contrast, activation of naive B cells from SLE patients expressed genes associated with activated memory B cells, such as Ab-sensing Fc receptor–like molecules. The authors showed that exposing B cells from SLE patients to IL-4 in vitro promoted their differentiation to plasmablasts and classical memory B cells. This study indicates that therapies promoting IL-4 signaling in B cells may alleviate SLE in patients with a high type I IFN signature.

In this Top Read, Dogan et al. (p. 1523) demonstrated that mucosal-associated invariant T (MAIT) cells can be engineered with chimeric Ag receptors (CARs) to effectively target cancer cells. The authors engineered CAR-MAIT cells to express either anti-CD19 or anti-Her2, for targeting B cell lymphoma or breast cancers, respectively. Compared to CAR CD8+ T cells, CAR-MAIT cells showed greater cytotoxicity toward primary B cells and a breast cancer cell line, as shown by decreased E:T ratios in vitro. The cytokine profiles of CAR-MAIT cells were characterized by decreased IFN-γ and increased TNF in comparison with CAR CD8+ T cells. Unlike CAR CD8+ T cells, CAR-MAIT cells recognize MHC class I–related protein (MR1) that is presenting bacterial vitamin B2 metabolites, making them less likely to cause an autoimmune response. Indeed, primary MAIT cells treated with vitamin B metabolite 5-ARU showed dose-dependent cytotoxicity to MRI-expressing cancer lines. These data show that MAIT cells have the potential to be engineered into potent cancer immunotherapies.

In this Top Read, Cronk et al. (p. 1545) investigated how NK cells sense murine CMV (MCMV)-infected targets. It was previously shown that the MCMV protein m04/gp34 promotes immune evasion by escorting MHC class I (MHC I) molecules to the surface of infected cells where they bind to NK cell Ly49 inhibitory receptors, such as Ly49A. In this study, the authors showed that MCMV triggers NK viral sensing via the Ly49R activation receptor on Ly49G2-licenced NK cells, and that the antiviral sensing mechanism is dependent on MHCI Dk. Mechanistically, MCMV gp34 promotes Ly49G2 binding to infected cells, which is required for the antiviral effects of Ly49R. Finally, the authors found that Ly49G2 can license IL-12 signaling and promote proliferation in Ly49R+ NK cells. This study further elucidates the complex mechanism of antiviral NK activation and inhibitory receptor interactions during MCMV infection.