Qualified Biolayer Interferometry Avidity Measurements Distinguish the Heterogeneity of Antibody Interactions with Plasmodium falciparum Circumsporozoite Protein Antigens [NOVEL IMMUNOLOGICAL METHODS]

Ab avidity is a measure of the overall strength of Ab–Ag interactions and hence is important for understanding the functional efficiency of Abs. In vaccine evaluations, Ab avidity measurements can provide insights into immune correlates of protection and generate hypotheses regarding mechanisms of protection to improve vaccine design to achieve higher levels of efficacy. The commonly used Ab avidity assays require the use of chaotropic reagents to measure avidity index. In this study, using real-time detection of Ab–Ag binding by biolayer interferometry (BLI) technique, we have developed a qualified assay for measuring avidity of vaccine-induced Abs specific for Plasmodium falciparum circumsporozoite protein (CSP) Ags. Human mAb derived from plasmablasts of recipients of RTS,S/AS01 (RTS,S), the most advanced malaria vaccine candidate, were used in the assay development to measure Ag-specific binding responses and rate constants of association and dissociation. The optimized BLI binding assay demonstrated 1) good precision (percentage of coefficient of variation <20), 2) high specificity, 3) a lower limit of detection and quantitation in the 0.3–3.3 nM range, and 4) a range of linearity up to 50–100 nM for the CSP Ags tested. Analysis of polyclonal sera of malaria vaccinees demonstrated the suitability of this method to distinguish among vaccinees and rank Ab responses by avidity. These results demonstrate that precise, specific, and sensitive BLI measurements of Ab avidity in polyclonal sera from malaria vaccinees can map Ab response heterogeneity and potentially help to determine the role of Ab avidity as an immune correlate of protection for vaccines.

https://ift.tt/2OhhxRI

Tumor Dendritic Cells (DCs) Derived from Precursors of Conventional DCs Are Dispensable for Intratumor CTL Responses [TUMOR IMMUNOLOGY]

The success of adoptive CTL therapy for cancer depends on interactions between tumor-infiltrating CTLs and cancer cells as well as other cells and molecules in the tumor microenvironment. Tumor dendritic cells (DCs) comprise several subsets: CD103⁺CD11b^– DC1 and CD11b⁺CD64^– DC2, which originate from circulating precursors of conventional DCs, and CD11b⁺CD64⁺ DC3, which arise from monocytes. It remains controversial which of these subset(s) promotes intratumor CTL proliferation, expansion, and function. To address this issue, we used the Zbtb46-DTR–transgenic mouse model to selectively deplete DC1 and DC2 from tumors and lymphoid tissues. Wild-type and Zbtb46-DTR bone marrow chimeras were inoculated with B16 melanoma cells that express OVA and were treated with OT-1 CTLs. We found that depletion of DCs derived from precursors of conventional DCs in Zbtb46-DTR bone marrow chimeras abolished CTL proliferation and expansion in tumor-draining lymph nodes. By contrast, intratumor CTL accumulation, proliferation, and IFN- expression were unaffected by their absence. We found that adoptive cell therapy increases the frequency of monocyte-derived tumor DC3, which possess the capacity to cross-present tumor Ags and induce CTL proliferation. Our findings support the specialized roles of different DC subsets in the regulation of antitumor CTL responses.

https://ift.tt/2LXvGah

Variation in Genome-Wide NF-{kappa}B RELA Binding Sites upon Microbial Stimuli and Identification of a Virus Response Profile [SYSTEMS IMMUNOLOGY]

NF-B transcription factors are master regulators of the innate immune response. Activated downstream of pathogen recognition receptors, they regulate the expression of genes to help fight infections as well as recruit the adaptive immune system. NF-B responds to a wide variety of signals, but the processes by which stimulus specificity is attained remain unclear. In this article, we characterized the response of one NF-B member, RELA, to four stimuli mimicking infection in human nasopharyngeal epithelial cells. Comparing genome-wide RELA binding, we observed stimulus-specific sites, although most sites overlapped across stimuli. Specifically, the response to poly I:C (mimicking viral dsRNA and signaling through TLR3) induced a distinct RELA profile, binding in the vicinity of antiviral genes and correlating with corresponding gene expression. This group of binding sites was also enriched in IFN regulatory factor motifs and showed overlapping with IFN regulatory factor binding sites. A novel NF-B target, OASL, was further validated and showed TLR3-specific activation. This work showed that some RELA DNA binding sites varied in activation response following different stimulations and that interaction with more specialized factors could help achieve this stimulus-specific activity. Our data provide a genomic view of regulated host response to different pathogen stimuli.

https://ift.tt/2OhhzJk

CX3CR1+ Macrophages and CD8+ T Cells Control Intestinal IgA Production [MUCOSAL IMMUNOLOGY]

Secretory IgA is a key host defense mechanism that controls the intestinal microbiota. We investigated the role of CD11c⁺CX₃CR1⁺CD64⁺ macrophages in IgA production in the intestine. Intestinal CX₃CR1⁺ macrophages directly induced IgA secretion by B cells. Ag delivery to lamina propria (LP) CX₃CR1⁺ macrophages specifically induced intestinal IgA production. The induction of IgA by CX₃CR1⁺ macrophages required BAFF, a proliferation-inducing ligand, and TNF-α, but was surprisingly independent of TLR-mediated microbial recognition and retinoic acid signaling. IgA secretion by CX₃CR1⁺ macrophages was enhanced by LP CD8⁺ T cells through the secretion of IL-9 and IL-13. CX₃CR1⁺ macrophages and CD8⁺ T cells induced IgA production by B cells independently of mesenteric lymph nodes and Peyer patches. Our data reveal a previously unrecognized cellular circuitry in which LP CX₃CR1⁺ macrophages, B cells, and CD8⁺ T cells coordinate the protective Ig secretion in the small intestine upon peripheral Ag delivery.

https://ift.tt/2LXTJG2

Cellular Mechanisms Controlling Surfacing of AICL Glycoproteins, Cognate Ligands of the Activating NK Receptor NKp80 [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

AICL glycoproteins are cognate activation-induced ligands of the C-type lectin-like receptor NKp80, which is expressed on virtually all mature human NK cells, and NKp80–AICL interaction stimulates NK cell effector functions such as cytotoxicity and cytokine secretion. Notably, AICL and NKp80 are encoded by adjacent genes in the NK gene complex and are coexpressed by human NK cells. Whereas AICL is intracellularly retained in resting NK cells, exposure of NK cells to proinflammatory cytokines results in AICL surfacing and susceptibility to NKp80-mediated NK fratricide. In this study, we characterize molecular determinants of AICL glycoproteins that cause intracellular retention, thereby controlling AICL surface expression. Cys⁸⁷ residing within the C-type lectin-like domain not only ensures stable homodimerization of AICL glycoproteins by disulfide bonding, but Cys⁸⁷ is also required for efficient cell surface expression of AICL homodimers and essential for AICL–NKp80 interaction. In contrast, cytoplasmic lysines act as negative regulators targeting AICL for proteasomal degradation. One atypical and three conventional N-linked glycosylation sites in the AICL C-type lectin-like domain critically impact maturation and surfacing of AICL, which is strictly dependent on glycosylation of at least one conventional glycosylation site. However, although the extent of conventional N-linked glycosylation positively correlates with AICL surface expression, the atypical glycosylation site impairs AICL surfacing. Stringent control of AICL surface expression by glycosylation is reflected by the pronounced interaction of AICL with calnexin and the impaired AICL expression in calnexin-deficient cells. Collectively, our data demonstrate that AICL expression and surfacing are tightly controlled by several independent cellular posttranslational mechanisms.

https://ift.tt/2Od6mJQ

Glucocorticoids Inhibit Group 3 Innate Lymphocyte IL-22 Production [INNATE IMMUNITY AND INFLAMMATION]

Glucocorticoids (GCs) are commonly prescribed to patients with a variety of inflammatory disorders, including inflammatory bowel disease (IBD). GCs mediate their immunomodulatory effects through many different mechanisms and target multiple signaling pathways. The GC dexamethasone downmodulates innate and adaptive immune cell activation. IBD is the manifestation of a dysregulated immune response involving many different immune cells. Group 3 innate lymphocytes (ILC3s) have critical roles in mucosal inflammation. ILC3s secrete high levels of the cytokine IL-22, promoting epithelial proliferation, antimicrobial peptides, and mucins. In this study, we examined the effects of dexamethasone on IL-22 production by ILC3s. We found that dexamethasone suppressed IL-23–mediated IL-22 production in human and mouse ILC3s. This was mediated in part through dexamethasone modulation of the NF-B pathway. Inhibition of NF-B signaling with a small molecule inhibitor also downmodulated IL-23– and IL-1β–mediated IL-22 production in ILC3s. These findings implicate NF-B as a regulator of IL-22 in ILC3s and likely have repercussions on GC treatment of IBD patients.

https://ift.tt/2neNNcR

CD4+ Regulatory T Cells Exert Differential Functions during Early and Late Stages of the Immune Response to Respiratory Viruses [INFECTIOUS DISEASE AND HOST RESPONSE]

Acute respiratory virus infection (ARI) induces CD8⁺ T cells with diminished cytokine production and functional impairment. The role of cellular mediators of immune impairment, specifically CD4⁺ regulatory T cells (Tregs), is incompletely understood in ARI. Tregs are known suppressors of effector T cell function, but whether they are detrimental or beneficial in ARI remains controversial. We show in this paper that Treg depletion leads to increased CD8⁺ T cell function and lower virus titer in mice infected with human metapneumovirus. We further demonstrate that Tregs play a temporal role in the immune response to human metapneumovirus and influenza: Treg depletion before infection pathologically reduces virus-specific CD8⁺ T cell numbers and delays virus clearance, whereas depletion 2 d postinoculation enhances CD8⁺ T cell functionality without reducing virus-specific CD8⁺ T cell numbers. Mechanistically, Treg depletion during immune priming led to impaired dendritic cell and CD8⁺ T cell migration. Further, early Treg depletion was associated with immune skewing toward a type 2 phenotype characterized by increased type 2 innate lymphoid cells and T_H2 CD4⁺ T cells, which was not observed when Treg depletion was delayed until after inoculation. These results indicate that the presence of Tregs at inoculation is critical for efficient priming of the CD8⁺ T cell response to ARI, whereas later in infection, Tregs are dispensable for virus clearance.

https://ift.tt/2OdfAWz