IκBζ Augments IL-33–Driven Cytokine Production See article p. 2033

Harnessing MHC-E–Restricted CD8+ T Cells for Vaccines See article p. 2169

Type I IFN Protects Infant Mice during B. pertussis Infection See article p. 2192

Perforin-2: More than Meets the Eye See article p. 2242

Role of LIF Receptor in pDCs See article p. 2257

Gene Editing in Adoptively Transferred Naive T Cells See article p. 2308

In this Top Read, Sesti-Costa et al. (p. 2257) demonstrate that LIF inhibits plasmacytoid dendritic cell (pDC) development and activation. Unlike conventional DCs (cDCs), pDCs expressed high levels of functional LIF receptor (LIFR), which was downregulated upon CpG stimulation. LIF treatment inhibited pDC production of IFN-α, TNF, and IL-6 in response to CpG, reduced expression of IFN-α genes, and upregulated expression of Id2, a repressor of transcription factor E2-2, which is essential for pDC development. The authors demonstrated that LIFR was not expressed on common DC progenitors, but was detected on CD11c+ post–common DC progenitors, suggesting that LIF may bias late stage DC differentiation, post–common DC progenitor. LIF exposure promoted differentiation of CD11b+ cDC at the expense of pDC in bone marrow cultures, but in vivo development of DC was unaffected in mice lacking DC-specific LIFR expression. However, infection of mice lacking pDC-specific LIFR expression with lymphocytic choriomeningitis virus resulted in higher IFN-α serum levels, further suggesting that LIF signaling suppresses pDC IFN-α responses. Together, these data provide a novel pathway in pDCs that may be exploited to prevent inappropriate IFN-α secretion in the setting of autoimmunity or may be harnessed to promote IFN-α production during viral infections.

The IL-33 receptor, ST2L, is abundantly expressed in tissue-resident immune cells. In this Top Read, Ohto-Ozaki et al. (p. 2033) examined the role of the downstream signaling molecule IκBζ in ST2L-mediated cytokine and chemokine production in mast cells. Treatment of bone marrow–derived mast cells (BMMCs) with IL-33 induced robust expression of IκBζ and increased production of IL-6, IL-13, CCL2, and CCL3, as well as TNF-α protein and mRNA, whereas deletion of IκBζ reduced expression of these mediators. Examination of signaling pathways reportedly activated by IκBζ revealed that NF-κB p50, but not p65, translocated to the nucleus of IL-33–stimulated BMMCs, and this movement was not impacted by deletion of IκBζ. Additionally, inhibition of NF-κB significantly attenuated induction of IκBζ and pursuant cytokine and chemokine production. Deletion of IκBζ did not impact IL-33–induced phosphorylation of the mitogen-activated kinases ERK, p38 MAPK, or JNK, which are reportedly activated by IL-33 signaling; nor did it abolish the expression of the IκBζ gene. Collectively, this study demonstrates that IκBζ augments IL-33–dependent cytokine and chemokine production in mast cells via the action of NF-κB.

In this Top Read, Ardanuy et al. (p. 2192) demonstrate the potential use of type I and III IFNs as therapeutics during Bordetella pertussis infection in infants. In B. pertussis–infected adult mice, upregulation of lung type I IFN correlated with an increase in other proinflammatory cytokines, indicating a possible link between type I IFN and lung inflammation. Enhanced type I IFN signaling was correlated with an increase in lung inflammation during B. pertussis infection in adults, whereas knockout (KO) of the type I IFN receptor (IFNAR1) did not reduce lung inflammation. However, IFNAR1 KO mice had a significant increase in IFN-λ gene expression and treatment of IFNAR1 KO mice with neutralizing anti–IFN-λ Abs reduced lung inflammation, suggesting that IFNλ can compensate for the loss of type 1 IFN signaling. Infection of mice lacking the IFN-λ receptor with B. pertussis decreased lung inflammation. Wild-type (WT) infant mice did not upregulate type I or IFN-γ in response to B. pertussis infection. However, transgenic infant mice with enhanced type I IFN signaling exhibited lower inflammatory cytokine gene expression in response to B. pertussis infection, resulting in significantly reduced mortality when compared with WT mice. Together, these data provide a new role for IFN-λ during B. pertussis infection in adult mice and demonstrate that type I IFN mediates a protective effect against B. pertussis infection in neonates.

To date, gene editing strategies using CRISPR/Cas9 technologies have required in vitro stimulation or ex vivo culture of naive T cells, both of which alter in vivo differentiation and preclude studies of homeostasis. In this Top Read, Nüssing et al. (p. 2308) demonstrated highly efficient gene editing in uncultured, naive CD8+ T cells from mice by electroporation of recombinant Cas9/single guide RNA ribonucleoprotein immediately prior to adoptive transfer. In two different infection models, chronic lymphocytic choriomeningitis virus Clone 13 and OVA-transgenic Listeria monocytogenes, transferred cells underwent clonal expansion postinfection and lost target proteins at high efficiency. Gene deletion occurred in electroporated cells even when the cells were left in a naive state, suggesting that the Cas9-directed mutations occur independently of T cell activation. Finally, the authors showed that targeted, single nucleotide polymorphic mutations could be introduced into naive CD8+ T cells using CRISPR/Cas9-based homology-directed repair. Thus, the authors introduce a method that expands CRISPR-based editing techniques beyond approaches requiring robust T cell activation and that facilitates the study of T cell homeostasis and models of weak activation, such as tolerance and tumor immunity.

Although type I IFNs play a critical role during microbial infection, the mechanisms governing activation of IFN signaling are not well understood. In this Top Read, McCormack et al. (p. 2242) sought to elucidate the role Perforin-2 plays in regulating assembly of the IFN receptor complex. Mice deficient in Perforin-2 were resistant to LPS-induced septic shock and displayed impaired type I IFN–mediated JAK–STAT signaling, as well as delays in the activation of MAPK and PI3K signaling pathways. Furthermore, Perforin-2 deficiency resulted in defective type I–mediated STAT1 DNA binding activity and induction of antiviral genes, suggesting an essential role for Perforin-2 in type I IFN signaling. Perforin-2 localized primarily on the cell surface of bone marrow–derived macrophages and directly associated with IFN-α and -β receptors 1 and 2 (IFNAR1 and IFNAR2). Association was mediated by interactions of IFNAR1 and IFNAR2 with the extracellular N-glycosylated extracellular membrane attack complex (MACPF) and P2 domains, respectively. Additionally, the Perforin-2 cytoplasmic tail domain was found to regulate Jak1 and Tyk2 interactions via association with STAT2 and IFNAR2, which are critical for Jak1/Tyk2 reciprocal transphosphorylation and STAT activation upon IFN-β stimulation. Together, these data demonstrate that Perforin-2 is a regulator of IFNAR interactions and receptor proximal complex assembly during type I IFN signaling.

Major histocompatibility complex E–restricted CD8+ T cells are not naturally induced by hepatitis B virus (HBV) infection and are, therefore, not subject to exhaustion or tolerance during chronic hepatitis B virus infection (CHB). In this Top Read, Burwitz et al. (p. 2169) used recombinant vaccine vectors to elicit MHC-E–restricted CD8+ T cells as a potential therapy to treat CHB infection. Rhesus macaques (RM) produced robust CD8+ T cell responses following vaccination with CMV vaccine vectors engineered to express HBV Ag. Blockade of Ag presentation by MHC-I, MHC-II, and MHC-E showed that HBV-specific CD8+ T cells recognized cognate Ag in the context of MHC-E. Primary hepatocytes (PH) from both RM and human donors expressed MHC-E ex vivo. Following infection with HBV, PH from both species expressed moderate levels of surface MHC-E, indicating that MHC-E may represent an HBV-specific CD8+ T cell restriction element. CD8+ T cells from inoculated RM recognized HBV-infected PH. Blockade of MHC-E Ag presentation by PH eliminated the CD8+ T cell response, indicating that MHC-E was crucial for the HBV-specific CD8+ T cell response. Therefore, this proof-of-concept study demonstrates that MHC-E–restricted HBV Ag can be used to induce a CD8+ T cell response with potential for combatting CHB infection.