The programmed death-1 (PD-1) costimulatory receptor inhibits T and B cell responses and plays a crucial role in peripheral tolerance. PD-1 has recently been shown to inhibit T cell responses during ...chronic viral infections such as HIV. In this study, we examined the role of PD-1 in infection with Mycobacterium tuberculosis, a common co-infection with HIV. PD-1–deficient mice showed dramatically reduced survival compared with wild-type mice. The lungs of the PD-1⁻ / ⁻ mice showed uncontrolled bacterial proliferation and focal necrotic areas with predominantly neutrophilic infiltrates, but a lower number of infiltrating T and B cells. Proinflammatory cytokines, such as TNF-α, IL-1, and especially IL-6 and IL-17 were significantly increased in the lung and sera of infected PD-1⁻ / ⁻ mice, consistent with an aberrant inflammation. Microarray analysis of the lungs infected with M. tuberculosis showed dramatic differences between PD-1⁻ / ⁻ and control mice. Using high-stringency analysis criteria (changes of twofold or greater), 367 transcripts of genes were differentially expressed between infected PD-1⁻ / ⁻ and wild-type mice, resulting in profoundly altered inflammatory responses with implications for both innate and adaptive immunity. Overall, our studies show that the PD-1 pathway is required to control excessive inflammatory responses after M. tuberculosis infection in the lungs.
Tumor necrosis factor (TNF) antagonists are increasingly used for the treatment of inflammatory and autoimmune diseases. Immunogenicity of these drugs poses therapeutic challenges such as therapeutic ...failure and adverse effects in a number of patients. Evaluation of clinical nonresponsiveness includes laboratory testing for drug concentrations and detecting the presence of antidrug antibodies.
This review provides an overview of the immunogenicity of TNF antagonists and testing methodologies currently available for measuring antidrug antibody response, which decreases treatment efficacy and may result in therapeutic failure. This review summarizes methodologies such as binding assays, including ELISA and HPLC-based homogenous mobility shift assay, as well as functional cell-based assays such as reporter gene assay. Furthermore, based on the laboratory findings of testing for antidrug antibody response, as well as serum drug concentrations, an algorithm is provided for interpretation, based on the current available literature and guidelines, which may aid in determining optimal therapy after treatment failure.
Laboratory testing methodologies for measuring serum concentrations of TNF inhibitors and antidrug antibodies are clinically available. These methods provide an evidence-based, personalized approach for the workup of patients showing treatment failure, which saves time and resources, and contributes to improved patient care.
Monoclonal antibody therapeutics (MATs) represent a rapidly expanding class of biological drugs used to treat a variety of diseases. The widespread use of MATs increasingly affects clinical ...laboratory medicine.
This review provides an overview of MATs currently approved for clinical use in the US, starting from basic biology of antibodies to the engineering, pharmacokinetic and pharmacodynamic properties, nomenclature, and production of MATs. Immunogenicity and the production of antidrug antibodies (ADAs) play a major role in loss of therapeutic response and the development of treatment failure to certain MATs. Laboratory-based monitoring for MATs and detection of ADAs represent emerging needs for optimizing the use of MATs to achieve the best outcomes at affordable cost. In addition, the increased use of MATs affects clinical laboratory testing by interference of MATs with clinical laboratory tests across different areas of laboratory medicine, including histocompatibility, blood bank, and monoclonal protein testing.
The number of MATs is rapidly growing each year to address previously unmet clinical needs. Laboratory monitoring of MATs and detecting ADAs represent expanding areas of laboratory testing. Test-based strategies allow for treatment optimization at the level of the individual patient, thus providing a personalized medicine approach. In addition, clinical laboratories must be aware that the increasing use of MATs affects laboratory testing and be ready to implement methods to eliminate or mitigate interference with clinical tests.
HLA associations have been linked to many diseases and are important for risk assessment of drug hypersensitivity reactions. The increasing number of HLA alleles discovered generated a list of ...ambiguities that cannot be resolved with the current clinical assays, which commonly include sequence-specific oligonucleotide probe (SSOP) genotyping, and real-time PCR with melting curve analysis. HLA typing by next-generation sequencing (NGS) has recently been adopted by clinical laboratories for transplantation testing, as it provides unambiguous and cost-effective HLA typing. The goal of this study was to evaluate the feasibility of using NGS-based HLA-B and DQ genotyping for clinical HLA disease association testing, and provide direct comparison with the currently used clinical tests, including SSOP genotyping, and real-time PCR with melting curve analysis. While the real-time PCR method is easy and inexpensive to perform, ambiguities are rapidly increasing as more and more HLA alleles are discovered. SSOP genotyping identifies the alleles present but limitations include ambiguities and underreporting less common alleles. Our data show that HLA typing by NGS is superior to the existing clinical methods for identifying HLA alleles associated with disease or drug hypersensitivity, and offers a viable approach for high volume clinical diagnostic laboratories.
Programmed death-1 (PD-1) is a member of the CD28/B7 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. The high-resolution crystal structure of the ...complex formed by the complete ectodomains of murine PD-1 and PD-L2 revealed a 1:1 receptor:ligand stoichiometry and displayed a binding interface and overall molecular organization distinct from that observed in the CTLA-4/B7 inhibitory complexes. Furthermore, our structure also provides insights into the association between PD-1 and PD-L1 and highlights differences in the interfaces formed by the two PD-1 ligands (PD-Ls) Mutagenesis studies confirmed the details of the proposed PD-1/PD-L binding interfaces and allowed for the design of a mutant PD-1 receptor with enhanced affinity. These studies define spatial and organizational constraints that control the localization and signaling of PD-1/PD-L complexes within the immunological synapse and provide a basis for manipulating the PD-1 pathways for immunotherapy.
Introduction
Since the 2018 change in the US adult heart allocation policy, more patients are bridged‐to‐transplant on temporary mechanical circulatory support (tMCS). Previous studies indicate that ...durable left ventricular assist devices (LVAD) may lead to allosensitization. The goal of this study was to assess whether tMCS implantation is associated with changes in sensitization.
Methods
We included patients evaluated for heart transplants between 2015 and 2022 who had alloantibody measured before and after MCS implantation. Allosensitization was defined as development of new alloantibodies after tMCS implant.
Results
A total of 41 patients received tMCS before transplant. Nine (22.0%) patients developed alloantibodies following tMCS implantation: 3 (12.0%) in the intra‐aortic balloon pump group (n = 25), 2 (28.6%) in the microaxial percutaneous LVAD group (n = 7), and 4 (44.4%) in the veno‐arterial extra‐corporeal membrane oxygenation group (n = 9)—p = .039. Sensitized patients were younger (44.7 ± 11.6 years vs. 54.3 ± 12.5 years, p = .044), were more likely to be sensitized at baseline ‐ 4 of 9 (44.4%) compared to 1 out of 32 (3.1%) (p = .001) and received more transfusions with red blood cells (6 (66.7%) vs. 8 (25%), p = .02) and platelets (6 (66.7%) vs. 5 (15.6%), p = .002). There was no significant difference in tMCS median duration of support (4 3,15 days vs. 8.5 5,14.5 days, p = .57). Importantly, out of the 11 patients who received a durable LVAD after tMCS, 5 (45.5%) became sensitized, compared to 4 out of 30 patients (13.3%) who only had tMCS—p = .028.
Conclusions
Our findings suggest that patients bridged‐to‐transplant with tMCS, without significant blood product transfusions and a subsequent durable LVAD implant, have a low risk of allosensitization. Further studies are needed to confirm our findings and determine whether risk of sensitization varies by type of tMCS and duration of support.
TNF-α antagonists such as infliximab are effective for the treatment of inflammatory bowel disease and other inflammatory and autoimmune diseases. Development of an immune response and subsequent ...neutralizing antibodies against these protein-based drugs is a major impediment that contributes to therapeutic failure, or adverse effects such as hypersensitivity reactions. As opposed to empirical dose-escalation strategies, rational and cost-effective evaluation of clinical non-responsiveness includes measurement of serum drug levels, and detection of drug-specific antibodies. We present the validation and 2-y experience using a functional, cell-based reporter gene assay (RGA) developed for measuring the biological activity and antibody response to serum infliximab.
The RGA was used to test 4699 clinical specimens from patients suspected of therapeutic failure. In contrast to binding assays, which detect an overall antibody response, the RGA specifically detects those antibodies that have drug-neutralizing function, and thus, poses higher risk for therapeutic failure.
The RGA presented here is currently the only functional clinical test available to measure serum infliximab activity and neutralizing antibodies.
Due to its accuracy and precision, and suitability for high-throughput testing, this robust platform can be applied to any TNF-α antagonist, providing an invaluable tool for the clinical management of patients with treatment failure.
•TNF-α antagonists are widely used for the treatment of inflammatory and autoimmune diseases.•Immunogenicity of TNF-α inhibitors poses therapeutic challenges and results in clinical non-responsiveness in some patients.•Non-responsive patients should be screened for drug levels, and the presence of drug-specific antibodies.•A cell-based functional reporter gene assay (RGA) is validated and available for clinical use.•The RGA provides a precise and accurate way to measure serum drug activity, and detect drug-specific neutralizing antibodies.
T cell activity is controlled by a combination of antigen-dependent signaling through the T cell receptor and a set of auxiliary signals delivered through antigen-independent interactions, including ...the recognition of the B7 family of ligands. B7-H3 is a recently identified B7 family member that is strongly overexpressed in a range of cancers and correlates with poor prognosis. We report the crystal structure of murine B7-H3 at a 3 Å resolution, which provides a model for the organization of the IgV and IgC domains within the ectodomain. We demonstrate that B7-H3 inhibits T cell proliferation and show that the FG loop of the IgV domain plays a critical role in this function. B7-H3 crystallized as an unusual dimer arising from the exchange of the G strands in the IgV domains of partner molecules. This arrangement, in combination with previous reports, highlights the dynamic nature and plasticity of the immunoglobulin fold.
► Murine B7-H3 ectodomain crystals contain a strand-swapped dimer ► Recombinant B7-H3 ectodomain inhibits T cell proliferation ► Loop sequence mutations in the IgV domain disrupt B7-H3 dimerization and function
T cell activity is controlled by several distinct interactions, including those arising from members of the B7 protein family. Vigdorovich et al. report the crystal structure of murine B7-H3, which provides a model for the organization of the IgV and IgC domains within the ectodomain.
Costimulatory receptors and ligands trigger the signaling pathways that are responsible for modulating the strength, course, and duration of an immune response. High-resolution structures have ...provided invaluable mechanistic insights by defining the chemical and physical features underlying costimulatory receptor:ligand specificity, affinity, oligomeric state, and valency. Furthermore, these structures revealed general architectural features that are important for the integration of these interactions and their associated signaling pathways into overall cellular physiology. Recent technological advances in structural biology promise unprecedented opportunities for furthering our understanding of the structural features and mechanisms that govern costimulation. In this review, we highlight unique insights that have been revealed by structures of costimulatory molecules from the immunoglobulin and tumor necrosis factor superfamilies and describe a vision for future structural and mechanistic analysis of costimulation. This vision includes simple strategies for the selection of candidate molecules for structure determination and highlights the critical role of structure in the design of mutant costimulatory molecules for the generation of in vivo structure-function correlations in a mammalian model system. This integrated 'atoms-to-animals' paradigm provides a comprehensive approach for defining atomic and molecular mechanisms.
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