The primary cosignaling receptors belong to either the Ig CD28-like or TNF receptor (TNFR) superfamilies (1-5). Currently, the CD28 family consists of five lymphoid-specific coreceptors [CD28, inducible T cell costimulator (ICOS), cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death-1 (PD-1), and B and T lymphocyte attenuator (BTLA)] (1-3). CD28 and ICOS are single Ig-variable (IgV) domain glycoproteins that promote T cell activation, whereas the structurally related CTLA-4, PD-1, and BTLA receptors function to attenuate T cell activation. To date, all of the ligands that have been described for the CD28-like family members belong to the B7 superfamily (1-3, 6-9). Six B7 family members have been described, all of which have conserved extracellular IgV and Ig-constant domains (3). In the TNFR-TNF superfamily, five receptor-ligand interactions have been described that act as positive regulators. These include OX40-OX40L, 4-1BB-4-1BBL, CD27-CD70, CD30-CD30L, and herpesvirus entry mediator (HVEM)-homologous to lymphotoxin, which shows inducible expression and competes with herpes simplex virus (HSV) glycoprotein D (gD) for HVEM, a receptor expressed by T lymphocytes (HVEM-LIGHT) (4).The most recently identified CD28 family member is the inhibitory coreceptor, BTLA (10-12). BTLA is expressed on developing B and T cells, all mature lymphocytes, splenic macrophages, and mature marrow-derived dendritic cells (10,11). BTLA contains a single IgV domain and two intracellular immunoreceptor tyrosine-based inhibitory motifs that are phosphorylated after BTLA coligation to antigen receptors, resulting in recruitment of protein tyrosine phosphatases SHP-1 and SHP-2 (13). Because of this, and that coligation of BTLA to the TCR inhibits T cell activation, BTLA is implicated as a negative regulator of T cell activation (10, 11). This finding is further supported by the observation that BTLA-deficient T cells show increased proliferation and that BTLA Ϫ͞Ϫ mice have increased Ab response and show increased incidence and severity to an autoimmune disorder (9, 10).Initially, BTLA was proposed to interact with a B7 family member called B7x (10, 14). This conclusion, however, was based on an indirect binding study testing the interaction of B7x-Ig fusion to spleen and lymph node cells from either WT or BTLA-deficient mice. Furthermore, we have been unable to detect any specific binding of BTLA-Fc to B7x-transfected cells (data not shown). Spurred by the inability to confirm the BTLA-B7x interaction, we screened a secreted protein library (15) by using surface plasmon resonance (SPR) and identified HVEM as a coreceptor for human BTLA.Here we show that BTLA and HVEM interact with high affinity and can form a trimeric complex with TNF ligands LIGHT or lymphotoxin ␣ (LT␣). Our binding studies suggest that BTLA interacts with the outer surface of the HVEM͞TNF complex, suggesting structural models for how HVEM might engage BTLA on the cell surface. Finally, we demonstrate that binding of HVEM to BTLA results in the inhibition of T cel...

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkin's lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkin's lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for

Replicative aging of Saccharomyces cerevisiae is an established model system for eukaryotic cellular aging. A limitation in yeast lifespan studies has been the difficulty of separating old cells from young cells in large quantities. We engineered a new platform, the Miniature-chemostat Aging Device (MAD), that enables purification of aged cells at sufficient quantities for genomic and biochemical characterization of aging yeast populations. Using MAD, we measured DNA accessibility and gene expression changes in aging cells. Our data highlight an intimate connection between aging, growth rate, and stress. Stress-independent genes that change with age are highly enriched for targets of the signal recognition particle (SRP). Combining MAD with an improved ATAC-seq method, we find that increasing proteasome activity reduces rDNA instability usually observed in aging cells and, contrary to published findings, provide evidence that global nucleosome occupancy does not change significantly with age.

We present IDEA (the Induction Dynamics gene Expression Atlas), a dataset constructed by independently inducing hundreds of transcription factors (TFs) and measuring timecourses of the resulting gene expression responses in budding yeast. Each experiment captures a regulatory cascade connecting a single induced regulator to the genes it causally regulates. We discuss the regulatory cascade of a single TF, Aft1, in detail; however, IDEA contains > 200 TF induction experiments with 20 million individual observations and 100,000 signal‐containing dynamic responses. As an application of IDEA, we integrate all timecourses into a whole‐cell transcriptional model, which is used to predict and validate multiple new and underappreciated transcriptional regulators. We also find that the magnitudes of coefficients in this model are predictive of genetic interaction profile similarities. In addition to being a resource for exploring regulatory connectivity between TFs and their target genes, our modeling approach shows that combining rapid perturbations of individual genes with genome‐scale time‐series measurements is an effective strategy for elucidating gene regulatory networks.

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.