Certain microbes evade host innate immunity by killing activated macrophages with the help of virulence factors that target prosurvival pathways. For instance, infection of macrophages with the TLR4-activating bacterium Bacillus anthracis triggers an apoptotic response due to inhibition of p38 MAP kinase activation by the bacterial-produced lethal toxin. Other pathogens induce macrophage apoptosis by preventing activation of NF-kappaB, which depends on IkappaB kinase beta (IKKbeta). To better understand how p38 and NF-kappaB maintain macrophage survival, we searched for target genes whose products prevent TLR4-induced apoptosis and a p38-dependent transcription factor required for their induction. Here we describe key roles for transcription factor CREB, a target for p38 signaling, and the plasminogen activator 2 (PAI-2) gene, a target for CREB, in maintenance of macrophage survival.

The major histocompatibility complex (MHC) class II transactivator (CIITA) is the 'master coactivator' of MHC class II genes. To identify new targets of CIITA, we analyzed cDNA microarrays of dendritic cells (DCs) from CIITA-deficient, MHC class II-deficient and control mice. We found the semaphorin receptor plexin-A1 was expressed in DCs, but not in other immune cells, and was strongly induced by CIITA. RNA interference by short hairpin RNA specific for plexin-A1, but not a single-nucleotide mutant, greatly reduced plexin-A1 expression and T cell stimulation by protein- or peptide-antigen-pulsed DCs.Plexin-A1 is not required for peptide binding to MHC. These data indicate involvement of plexin-A1 in T cell-DC interactions but not antigen processing or binding.

Viruses have evolved elaborate mechanisms to target many aspects of the host's immune response. The cytokine IFN-gamma plays a central role in resistance of the host to infection via direct antiviral effects as well as modulation of the immune response. In this study, we demonstrate that the Epstein-Barr virus (EBV) immediate-early protein, BZLF1, inhibits the IFN-gamma signaling pathway. BZLF1 decreases the ability of IFN-gamma to activate a variety of important downstream target genes, such as IRF-1, p48, and CIITA, and prevents IFN-gamma-induced class II MHC surface expression. Additionally, BZLF1 inhibits IFN-gamma-induced STAT1 tyrosine phosphorylation and nuclear translocation. Finally, we demonstrate that BZLF1 decreases expression of the IFN-gamma receptor, suggesting a mechanism by which EBV may escape antiviral immune responses during primary infection.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Highlights d The immunoglobulin superfamily (IgSF) interactome identifies over 500 receptor pairs d The IgSF interactome reveals receptor-ligand communities dysregulated in cancer d More than 60 receptor-ligand pairs confirmed using orthogonal methods d Interacting protein signatures are correlated with clinical outcome