BATF3 programs CD8+ T cell memory.

Antiviral CD8+ T cell responses are characterized by an initial activation/priming of T lymphocytes followed by a massive proliferation, subset differentiation, population contraction and the development of a stable memory pool. The transcription factor BATF3 has been shown to play a central role in the development of conventional dendritic cells, which in turn are critical for optimal priming of CD8+ T cells. Here we show that BATF3 was expressed transiently within the first days after T cell priming and had long-lasting T cell-intrinsic effects. T cells that lacked Batf3 showed normal expansion and differentiation, yet succumbed to an aggravated contraction and had a diminished memory response. Vice versa, BATF3 overexpression in CD8+ T cells promoted their survival and transition to memory. Mechanistically, BATF3 regulated T cell apoptosis and longevity via the proapoptotic factor BIM. By programing CD8+ T cell survival and memory, BATF3 is a promising molecule to optimize adoptive T cell therapy in patients.

Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Overcoming key challenges in cancer immunotherapy with engineered T cells.

PURPOSE OF REVIEW: A number of clinical trials are currently testing chimeric antigen receptor (CAR) and T cell receptor (TCR) engineered T cells for the treatment of haematologic malignancies and selected solid tumours, and CD19-CAR-T cells have produced impressive clinical responses in B-cell malignancies. Here, we summarize the current state of the field, highlighting the key aspects required for the optimal application of CAR and TCR-engineered T cells for cancer immunotherapy. RECENT FINDINGS: Toxicities, treatment failure and disease recurrence have been observed at different rates and kinetics. Several strategies have been designed to overcome these hurdles: the identification and combination of known and new antigens, together with the combination of immunotherapeutic and classical approaches may overcome cancer immune evasion. New protocols for genetic modification and T cell culture may improve the overall fitness of cellular products and their resistance to hostile tumour immunomodulatory signals. Finally, the schedules of T cell administration and toxicity management have been adapted to improve the safety of this transformative therapeutic approach. SUMMARY: In order to develop effective adoptive T cell treatments for cancer, therapeutic optimization of engineered CAR and TCR T cells is crucial, by simultaneously focusing on intrinsic and extrinsic factors. This review focuses on the innovative approaches designed and tested to overcome the hurdles encountered so far in the clinical practice, with new excitement on novel laboratory insights and ongoing clinical investigations.

Cancer stratification by molecular imaging.

The lack of specificity of traditional cytotoxic drugs has triggered the development of anticancer agents that selectively address specific molecular targets. An intrinsic property of these specialized drugs is their limited applicability for specific patient subgroups. Consequently, the generation of information about tumor characteristics is the key to exploit the potential of these drugs. Currently, cancer stratification relies on three approaches: Gene expression analysis and cancer proteomics, immunohistochemistry and molecular imaging. In order to enable the precise localization of functionally expressed targets, molecular imaging combines highly selective biomarkers and intense signal sources. Thus, cancer stratification and localization are performed simultaneously. Many cancer types are characterized by altered receptor expression, such as somatostatin receptors, folate receptors or Her2 (human epidermal growth factor receptor 2). Similar correlations are also known for a multitude of transporters, such as glucose transporters, amino acid transporters or hNIS (human sodium iodide symporter), as well as cell specific proteins, such as the prostate specific membrane antigen, integrins, and CD20. This review provides a comprehensive description of the methods, targets and agents used in molecular imaging, to outline their application for cancer stratification. Emphasis is placed on radiotracers which are used to identify altered expression patterns of cancer associated markers.

New species based on the biological species concept within the complex of Lariophagus distinguendus (Hymenoptera, Chalcidoidea, Pteromalidae), a parasitoid of household pests.

The pteromalid parasitoid Lariophagus distinguendus (Foerster) belongs to the Hymenoptera, a megadiverse insect order with high cryptic diversity. It attacks stored product pest beetles in human storage facilities. Recently, it has been shown to consist of two separate species. To further study its cryptic diversity, strains were collected to compare their relatedness using barcoding and nuclear genes. Nuclear genes identified two clusters which agree with the known two species, whereas the barcode fragment determined an additional third Clade. Total reproductive isolation (RI) according to the biological species concept (BSC) was investigated in crossing experiments within and between clusters using representative strains. Sexual isolation exists between all studied pairs, increasing from slight to strong with genetic distance. Postzygotic barriers mostly affected hybrid males, pointing to Haldane's rule. Hybrid females were only affected by unidirectional Spiroplasma-induced cytoplasmic incompatibility and behavioural sterility, each in one specific strain combination. RI was virtually absent between strains separated by up to 2.8% COI difference, but strong or complete in three pairs from one Clade each, separated by at least 7.2%. Apparently, each of these clusters represents one separate species according to the BSC, highlighting cryptic diversity in direct vicinity to humans. In addition, these results challenge the recent 'turbo-taxonomy' practice of using 2% COI differences to delimitate species, especially within parasitic Hymenoptera. The gradual increase in number and strength of reproductive barriers between strains with increasing genetic distance also sheds light on the emergence of barriers during the speciation process in L. distinguendus.

The emergence of ecotypes in a parasitoid wasp: a case of incipient sympatric speciation in Hymenoptera?

BACKGROUND: To understand which reproductive barriers initiate speciation is a major question in evolutionary research. Despite their high species numbers and specific biology, there are only few studies on speciation in Hymenoptera. This study aims to identify very early reproductive barriers in a local, sympatric population of Nasonia vitripennis (Walker 1836), a hymenopterous parasitoid of fly pupae. We studied ecological barriers, sexual barriers, and the reduction in F1-female offspring as a postmating barrier, as well as the population structure using microsatellites. RESULTS: We found considerable inbreeding within female strains and a population structure with either three or five subpopulation clusters defined by microsatellites. In addition, there are two ecotypes, one parasitizing fly pupae in bird nests and the other on carrion. The nest ecotype is mainly formed from one of the microsatellite clusters, the two or four remaining microsatellite clusters form the carrion ecotype. There was slight sexual isolation and a reduction in F1-female offspring between inbreeding strains from the same microsatellite clusters and the same ecotypes. Strains from different microsatellite clusters are separated by a reduction in F1-female offspring. Ecotypes are separated only by ecological barriers. CONCLUSIONS: This is the first demonstration of very early reproductive barriers within a sympatric population of Hymenoptera. It demonstrates that sexual and premating barriers can precede ecological separation. This indicates the complexity of ecotype formation and highlights the general need for more studies within homogenous populations for the identification of the earliest barriers in the speciation process.

The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells.

Immunotherapy with chimeric antigen receptor (CAR)-engineered T cells can be effective against advanced malignancies. CAR T cells are "living drugs" that require technologies to enable physicians (and patients) to maintain control over the infused cell product. Here, we demonstrate that the tyrosine kinase inhibitor dasatinib interferes with the lymphocyte-specific protein tyrosine kinase (LCK) and thereby inhibits phosphorylation of CD3ζ and ζ-chain of T cell receptor-associated protein kinase 70 kDa (ZAP70), ablating signaling in CAR constructs containing either CD28_CD3ζ or 4-1BB_CD3ζ activation modules. As a consequence, dasatinib induces a function-off state in CD8+ and CD4+ CAR T cells that is of immediate onset and can be sustained for several days without affecting T cell viability. We show that treatment with dasatinib halts cytolytic activity, cytokine production, and proliferation of CAR T cells in vitro and in vivo. The dose of dasatinib can be titrated to achieve partial or complete inhibition of CAR T cell function. Upon discontinuation of dasatinib, the inhibitory effect is rapidly and completely reversed, and CAR T cells resume their antitumor function. The favorable pharmacodynamic attributes of dasatinib can be exploited to steer the activity of CAR T cells in "function-on-off-on" sequences in real time. In a mouse model of cytokine release syndrome (CRS), we demonstrated that a short treatment course of dasatinib, administered early after CAR T cell infusion, protects a proportion of mice from otherwise fatal CRS. Our data introduce dasatinib as a broadly applicable pharmacologic on/off switch for CAR T cells.

Molecular and cytogenetic differentiation within the Lariophagusdistinguendus (Förster, 1841) species complex (Hymenoptera, Pteromalidae).

Several strains of the apparently well-known cosmopolitan synanthropic parasitoid of coleopteran stored-product pests, Lariophagusdistinguendus (Förster, 1841) from Western Europe, were studied using DNA sequencing and chromosomal analysis. The presence of at least two cryptic species with different COI sequences and chromosome numbers (n = 5 and 6) was supported. The species with n = 6 is associated with the drugstore beetle Stegobiumpaniceum (Linnaeus, 1758), whereas the other one with n = 5 mostly develops on the granary weevil Sitophilusgranarius (Linnaeus, 1758). A phylogenetic study revealed that the karyotype with n = 6 represents an ancestral character state in this complex. Consequently, the chromosome set with n = 5 which is characteristic of a particular internal clade, apparently originated via chromosomal fusion which was probably preceded by a pericentric inversion. If this is true, inverted chromosome segments could accumulate a number of genetic loci responsible for certain interspecific differences.

From rabbit antibody repertoires to rabbit monoclonal antibodies.

In this review, we explain why and how rabbit monoclonal antibodies have become outstanding reagents for laboratory research and increasingly for diagnostic and therapeutic applications. Starting with the unique ontogeny of rabbit B cells that affords highly distinctive antibody repertoires rich in in vivo pruned binders of high diversity, affinity and specificity, we describe the generation of rabbit monoclonal antibodies by hybridoma technology, phage display and alternative methods, along with an account of successful humanization strategies.

A comparison study: paper-based versus web-based data collection and management.

The purpose of this pilot study was to compare the cost, accuracy, and efficiency of a web-enhanced handheld computer data collection system with those of the traditional paper-based data collection and management system and to increase awareness/knowledge of researchers on these two data collection and management methods. This is an important topic because funding resources are diminishing and high startup costs associated with automated data collection systems may give researchers pause when faced with these financial expenditures. Hence, this information will position grant writers and funders to make intelligent decisions regarding the feasibility and advantage of web-enhanced electronic data collection strategies.