Transmembrane domain-driven PD-1 dimers mediate T cell inhibition.

Programmed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease. Some immune receptors function as dimers, but PD-1 has been considered monomeric. Here, we show that PD-1 and its ligands form dimers as a consequence of transmembrane domain interactions and that propensity for dimerization correlates with the ability of PD-1 to inhibit immune responses, antitumor immunity, cytotoxic T cell function, and autoimmune tissue destruction. These observations contribute to our understanding of the PD-1 axis and how it can potentially be manipulated for improved treatment of cancer and autoimmune diseases.

The structural features that distinguish PD-L2 from PD-L1 emerged in placental mammals.

Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells. PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance to monitor real-time binding of recombinantly-expressed and -purified proteins, we found that W110PD-L2 acts as an "elbow" that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Furthermore, we identified a "latch" between the C and D ß-strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110PD-L2 "elbow" and a C-D region "latch." Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2-affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4ß7.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

The wide utility of rabbits as models of human diseases.

Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur's rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.

Imbalanced expression of Tif1γ inhibits pancreatic ductal epithelial cell growth.

Transcriptional intermediary factor 1 gamma (Tif1γ) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGFß pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1γ interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1γ inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1γ and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1γ resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1γ resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1γ reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1γ results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1γ may inhibit tumor invasion. These data suggest that Tif1γ might serve as a potential therapeutic target for pancreatic cancer.

The inhibition effect of anti-GPIIIa49-66 antibody on megakaryocyte differentiation.

We previously reported that patients with early-onset HIV-1 ITP developed a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cell for platelets, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5-fold in in vitro culture of human cord blood CD34+ cells driven by thrombopoietin (TPO). We also observe a three-fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody. However, we could not detect ROS release in DCFH-loaded mouse megakaryoblastic cells L8057 treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of L8057 cells induced by TPO. In fact, we found a dose dependent increase in the percentage of CD41 positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 cells treated with various concentrations of H2O2 (from 5 to 20 µM). We therefore conclude that the anti-GPIIIa49-66 antibody inhibits MK differentiation through ß3 integrin signalling independent of ROS release.

Androgen receptor coactivator p44/Mep50 in breast cancer growth and invasion.

Hormones and their receptors play an important role in the development and progression of breast carcinoma. Although the primary focus has been on oestrogen and oestrogen receptor (ER), androgen, androgen receptor (AR) and its coactivator(s) have been implicated in tumorigenesis of breast carcinoma and warrant further investigation. AR coactivator p44/Mep50 is identified as a subunit of methylosome complex and lately characterized as an AR coactivator that enhances AR mediated transcription activity in a ligand dependent manner. In prostate cancer, p44 is expressed in the nucleus of benign epithelia and translocated into the cytoplasm in cancer cells. Furthermore, nuclear expression of p44 inhibits prostate cancer growth. In this report, we examined the expression and function of p44 in breast cancer. In addition to being an AR coactivator, p44 also functions as an ER coactivator. In contrast to findings in prostate cancer, the expression of p44 shows strong cytoplasmic expression in morphologically normal terminal ductal lobular units, while nuclear p44 is observed in both ductal carcinoma in situ and invasive carcinoma. Further, overexpression of nuclear-localized p44 stimulates proliferation and invasion in MCF7 breast cancer cells in the presence of oestrogen and the process is ERα dependent. These findings strongly suggest that p44 plays a role in mediating the effects of hormones during tumorigenesis in breast.

C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates, and protects against carotid artery occlusion and cerebral stroke.

Anti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385-amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP or collagen and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti-ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCI3-induced carotid artery thrombosis as well as cerebral infarction in a postischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18.

A microtubule associated protein (hNUDC) binds to the extracellular domain of thrombopoietin receptor (Mpl).

Human NUDC (hNUDC) was initially characterized as a nuclear migration protein based on the similarity of its C-terminus to that of fungal NUDC from Aspergillus nidulans. However, hNUDC is a 331 amino acid protein whereas fungal NUDC is 198 amino acids in length. The extra N-terminal portion of hNUDC has no known function or homology to other proteins. In this study, we report the binding of hNUDC to the extracellular domain of the thrombopoietin receptor (Mpl) as detected by the yeast two-hybrid system, GST pull-down, and co-immunoprecipitation. Our deletion analysis demonstrated that amino acids between positions 100 and 238 as the critical domain mediating the hNUDC and Mpl interactions as detected by the two-hybrid system and GST pull-down assay. Immunofluorescence staining of human megakaryocyte cells indicated that hNUDC and Mpl colocalized at all stages of megakaryocyte development. Substantial colocalization of hNUDC with microtubules was also detected around nuclei and elongated microtubular structures, especially in proplatelet extensions.

Expression of the soluble extracellular domain of human thrombopoietin receptor using a maltose-binding protein-affinity fusion system.

The thrombopoietin (TPO) receptor (Mpl) belongs to the family of ligand-dependent cytokine receptors and plays a functional role in regulating platelet production. The signaling capacity largely depends on the binding of TPO to the extracellular domains of the TPO receptor (Mpl-EC). Because the expression level of Mpl in human tissue is very low, studies on the functional and spatial characteristics of its ligand-binding sites have been limited. In the present study, we report the expression and purification of Mpl-EC as a fusion with the maltose-binding protein (MBP), designated MBP-Mpl-EC. MBP-Mpl-EC was expressed in the cytoplasm of Escherichia coli as a soluble fusion protein. Specific binding of TPO to purified MBP-Mpl-EC was demonstrated by a dot-blot assay and surface plasmon resonance. We conclude that bacterial expression of MBP-Mpl-EC yields large amounts of protein with correct folding and that it can be used for further structure and function analyses.