Creating Broadly Neutralizing Abs against Influenza See article p. 375

Regulation of Nlrp3 by Micropeptides See article p. 428

Fibrinogen-Triggered Degranulation of Eosinophils See article p. 438

LSD1 Regulates PD-1 Expression See article p. 449

This Top Read features evidence that computationally optimized broadly reactive Ags (COBRA) in recombinant viruses can produce broadly neutralizing Abs against influenza hemagglutinin (HA). The COBRA methodology relies on multiple rounds of consensus sequence generation to design highly immunogenic and cross-reactive epitopes. In this study, Sautto et al. (p. 375) investigate the quality of Abs generated in response to a COBRA recombinant influenza virus (P1) when compared with commonly used influenza vaccines. ELISpot analysis showed that immunization with P1 increased the breadth of recombinant HA (rHA)-binding Ab secreting cells compared with historical seasonal or pandemic vaccine strains. Of the 28 characterized mAbs elicited by vaccination with P1, 20% bound all rHA tested. Compared with the wild-type (WT) viruses that did not exhibit broad HA inhibition (HAI) activity against unrelated strains, nine mAbs possessed HAI activity against both P1 and pandemic viruses. One mAb, 1F8, also recognized an overlapping, but distinct, epitope compared with other narrowly HAI-positive mAbs elicited by either PI or WT virus. Sequence analysis showed that P1-elicited mAbs were encoded by distinct VH and VL segment rearrangements and possessed unique CDR3 sequences. These data support the use of COBRA in developing next-generation influenza vaccines to elicit influenza-specific broadly neutralizing Abs.

Previous studies have indicated that functional peptides encoded by short open reading frames (sORFs) of long noncoding RNAs (lncRNAs) may act as positive or negative regulators of inflammatory gene expression. In this Top Read, Bhatta et al. (p. 428) identified lncRNA 1810058I24Rik, which was downregulated in both murine and human myeloid cells stimulated with TLR ligands and inflammatory cytokines. lncRNA 1810058I24Rik was located primarily in the cytosol and encoded a 47 aa micropeptide that was endogenously expressed on the mitochondrion of macrophages and was thus named mitochondrial micropeptide-47 (Mm47). Deletion of Mm47 in murine primary macrophages had no impact on signaling events downstream of TLR4 activation. In contrast, Mm47 was required for Nlrp3-dependent IL-1β maturation. The impact of Mm47 on inflammasome signaling was highly specific, as deletion of Mm47 had no impact on the activation of Nlrc4 or Aim2 inflammasomes. Therefore, this study identifies a novel mitochondrial micropeptide that is required for the activation of Nlrp3 and highlights the importance of sORF-encoded peptides in regulating innate immune responses.

During chronic exposure to Ags, programmed cell death–1 (PD-1) remains stably expressed across generations of T cell clones, suggesting epigenetic regulation of the Pdcd1 locus. In this Top Read, Bally et al. (p. 449) sought to further elucidate the epigenetic mechanisms regulating PD-1 expression. The authors observed changes in histone H3K4 methylation at the Pdcd1 locus during ex vivo and in vivo activation of CD8 T cells, suggesting a role for the histone H3 lysine 4 demethylase LSD1 in regulating PD-1 expression. Consistent with these observations, CD8 T cells lacking LSD1 expressed higher levels of Pdcd1 mRNA following ex vivo stimulation, as well as increased surface levels of PD-1 during acute, but not chronic lymphocytic choriomeningitis virus (LCMV) infection. Additionally, B lymphocyte–induced maturation protein–1 (Blimp-1), a known repressor of PD-1, recruited LSD1 to the Pdcd1 gene during acute, but not chronic, infection. Loss of DNA methylation at the promoter-proximal regulatory regions of Pdcd1 correlated with PD-1 expression. However, following acute LCMV infection, when PD-1 expression levels return to baseline, LSD1-deficient CD8 T cells failed to remethylate the Pdcd1 locus to wild-type levels. Finally, studies in a murine model of melanoma demonstrated that LSD1-deficient animals had a higher frequency of PD-1 expressing tumor-infiltrating lymphocytes. Thus, LSD1 is recruited to the Pdcd1 locus by Blimp-1 and downregulates PD-1 expression by removing the activating histone marks.

In this Top Reads Coden et al. (p. 438) demonstrate that fibrinogen is a specific trigger for cytolytic eosinophil degranulation. Fibrinogen is a complex glycoprotein with regulatory functions in many immune driven processes. When compared with eosinophils exposed to collagen, fibrinogen induced eosinophil cytolytic degranulation and upregulation of CD11b. Blockade of CD11b on eosinophils prior to fibrinogen exposure reduced adhesion, degranulation, and fibrinogen breakdown, suggesting that CD11b plays a role in the fibrinogen-dependent degranulation of eosinophils. Genetic analysis revealed that eosinophils exposed to fibrinogen downregulated genes associated with primary metabolic processes and cell migration, and significantly upregulated expression of inflammatory genes. Together, these data show that fibrinogen can directly trigger eosinophil degranulation, resulting in the degradation of fibrinogen, and provide a mechanism by which eosinophilic granules are deposited at sites of inflammation.