Using CombiCells, a platform for titration and combinatorial display of cell surface ligands, to study T-cell antigen sensitivity modulation by accessory receptors.

Understanding cellular decisions due to receptor-ligand interactions at cell-cell interfaces has been hampered by the difficulty of independently varying the surface density of multiple different ligands. Here, we express the synthetic binder protein SpyCatcher, designed to form spontaneous covalent bonds with interactors carrying a Spytag, on the cell surface. Using this, we show that addition of different concentrations and combinations of native Spytag-fused ligands allows for the combinatorial display of ligands on cells within minutes. We use this combinatorial display of cell surface ligands-called CombiCells-to assess T cell antigen sensitivity and the impact of T cell co-stimulation and co-inhibition receptors. We find that the T cell receptor (TCR) displayed greater sensitivity to peptides on major-histocompatibility complexes (pMHC) than synthetic chimeric antigen receptor (CARs) and bi-specific T cell engager (BiTEs) display to their target antigen, CD19. While TCR sensitivity was greatly enhanced by CD2/CD58 interactions, CAR sensitivity was primarily but more modestly enhanced by LFA-1/ICAM-1 interactions. Lastly, we show that PD-1/PD-L1 engagement inhibited T cell activation triggered solely by TCR/pMHC interactions, as well as the amplified activation induced by CD2 and CD28 co-stimulation. The ability to easily produce cells with different concentrations and combinations of ligands should accelerate the study of receptor-ligand interactions at cell-cell interfaces.

Interleukin-10 Directly Inhibits CD8+ T Cell Function by Enhancing N-Glycan Branching to Decrease Antigen Sensitivity.

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity of CD8+ T cells was decreased during chronic infection and that this was directly mediated by IL-10. Mechanistically, we showed that IL-10 induced the expression of Mgat5, a glycosyltransferase that enhances N-glycan branching on surface glycoproteins. Increased N-glycan branching on CD8+ T cells promoted the formation of a galectin 3-mediated membrane lattice, which restricted the interaction of key glycoproteins, ultimately increasing the antigenic threshold required for T cell activation. Our study identified a regulatory loop in which IL-10 directly restricts CD8+ T cell activation and function through modification of cell surface glycosylation allowing the establishment of chronic infection.

Inefficient exploitation of accessory receptors reduces the sensitivity of chimeric antigen receptors.

Chimeric antigen receptors (CARs) can redirect T cells to target abnormal cells, but their activity is limited by a profound defect in antigen sensitivity, the source of which remains unclear. Here, we show that CARs have a > 100-fold lower antigen sensitivity compared to the T cell receptor (TCR) when antigen is presented on antigen-presenting cells (APCs) but nearly identical sensitivity when antigen is presented as purified protein. We next systematically measured the impact of engaging important T cell accessory receptors (CD2, LFA-1, CD28, CD27, and 4-1BB) on antigen sensitivity by adding their purified ligands. Unexpectedly, we found that engaging CD2 or LFA-1 improved the antigen sensitivity of the TCR by 125- and 22-fold, respectively, but improved CAR sensitivity by only < 5-fold. This differential effect of CD2 and LFA-1 engagement on the TCR vs. CAR was confirmed using APCs. We found that sensitivity to antigen can be partially restored by fusing the CAR variable domains to the TCR CD3ε subunit (also known as a TRuC) and fully restored by exchanging the TCRαß variable domains for those of the CAR (also known as STAR or HIT). Importantly, these improvements in TRuC and STAR/HIT sensitivity can be predicted by their enhanced ability to exploit CD2 and LFA-1. These findings demonstrate that the CAR sensitivity defect is a result of their inefficient exploitation of accessory receptors and suggest approaches to increase sensitivity.

Performance evaluation of the MAGLUMI Hepatitis B virus surface antigen chemiluminescence immunoassay.

A highly sensitive and reliable Hepatitis B virus surface antigen (HBsAg) measurement is essential to universal screening, timely diagnosis, and management of Hepatitis B virus (HBV) infection. This study aimed to evaluate the performance of MAGLUMI HBsAg chemiluminescence immunoassay (CLIA). MAGLUMI HBsAg (CLIA) was compared against ARCHITECT HBsAg. 411 HBsAg positive samples, including different stages of infection, genotypes, subtypes, mutants, and 30 seroconversion panels were tested to evaluate diagnostic sensitivity. Diagnostic specificity was evaluated by testing 205 hospitalized samples and 5101 blood donor samples. Precision, limit of blank (LoB), limit of detection (LoD), and linearity were also verified. The diagnostic sensitivity of the MAGLUMI HBsAg (CLIA) was 100% with better seroconversion sensitivity than ARCHITECT HBsAg. The MAGLUMI HBsAg (CLIA) has optimal detection efficacy for HBV subgenotypes samples. The analytical sensitivity is 0.039 IU/mL. The initial diagnostic specificity is 99.63% on blood donors and 96.59% on hospitalized samples. The verification data demonstrated high repeatability, a LoB of 0.02 IU/mL, LoD of 0.05 IU/mL and an excellent linearity of 0.050-250 IU/mL (R2 = 0.9946). The MAGLUMI HBsAg (CLIA) is proved a highly sensitive and reliable assay with optimal subgenotype detection efficacy.

IgM-bearing B cell affinity subpopulations possess differential antigen sensitivity in rainbow trout.

The need for accurate assessments of in vitro generated antibody prompted examination of the effect of antigen on secreted antibody concentrations and affinities. It was found that the antigen concentrations commonly employed for in vitro stimulation were able to significantly compromise IgM titer and affinity estimates in rainbow trout. Specifically, IgM titers were underestimated with the high affinity antibodies being specifically blocked. To remedy this, pulsed antigen cultures were employed, and it was found to reveal more accurate IgM antibody titers and affinity estimates. Additionally, pulsed dose responses provided evidence that high antigen concentrations specifically suppressed high affinity B cell induction. Optimal expression of high affinity antibodies required exposure to lower concentrations of antigen. Each affinity subpopulation appeared to possess a graded sensitivity to each dose of antigen, revealing the complex dynamic for differential IgM-bearing B cell induction that is possible during a response. These results reveal not only the need for antigen removal prior to in vitro antibody secretion, but also the possible role of high zone immunological tolerance on IgM affinity maturation in rainbow trout.

Regulation of effector and memory CD8(+) T cell function by inflammatory cytokines.

Cells communicate with each other through the production and secretion of cytokines, which are integral to the host response to infection. Once recognized by specific cytokine receptors expressed on the cell surface, these exogenous signals direct the biological function of a cell in order to adapt to their microenvironment. CD8(+) T cells are critical immune cells that play an important role in the control and elimination of intracellular pathogens. Current findings have demonstrated that cytokines influence all aspects of the CD8(+) T cell response to infection or immunization. The cytokine milieu induced at the time of activation impacts the overall magnitude and function of the effector CD8(+) T cell response and the generation of functional memory CD8(+) T cells. This review will focus on the impact of inflammatory cytokines on different aspects of CD8(+) T cell biology.

Advanced Quantification of Receptor-Ligand Interaction Lifetimes via Single-Molecule FRET Microscopy.

Receptor-ligand interactions at cell interfaces initiate signaling cascades essential for cellular communication and effector functions. Specifically, T cell receptor (TCR) interactions with pathogen-derived peptides presented by the major histocompatibility complex (pMHC) molecules on antigen-presenting cells are crucial for T cell activation. The binding duration, or dwell time, of TCR-pMHC interactions correlates with downstream signaling efficacy, with strong agonists exhibiting longer lifetimes compared to weak agonists. Traditional surface plasmon resonance (SPR) methods quantify 3D affinity but lack cellular context and fail to account for factors like membrane fluctuations. In the recent years, single-molecule Förster resonance energy transfer (smFRET) has been applied to measure 2D binding kinetics of TCR-pMHC interactions in a cellular context. Here, we introduce a rigorous mathematical model based on survival analysis to determine exponentially distributed receptor-ligand interaction lifetimes, verified through simulated data. Additionally, we developed a comprehensive analysis pipeline to extract interaction lifetimes from raw microscopy images, demonstrating the model's accuracy and robustness across multiple TCR-pMHC pairs. Our new software suite automates data processing to enhance throughput and reduce bias. This methodology provides a refined tool for investigating T cell activation mechanisms, offering insights into immune response modulation.

Fine-tuning the antigen sensitivity of CAR T cells: emerging strategies and current challenges.

Chimeric antigen receptor (CAR) T cells are "living drugs" that specifically recognize their target antigen through an antibody-derived binding domain resulting in T cell activation, expansion, and destruction of cognate target cells. The FDA/EMA approval of CAR T cells for the treatment of B cell malignancies established CAR T cell therapy as an emerging pillar of modern immunotherapy. However, nearly every second patient undergoing CAR T cell therapy is suffering from disease relapse within the first two years which is thought to be due to downregulation or loss of the CAR target antigen on cancer cells, along with decreased functional capacities known as T cell exhaustion. Antigen downregulation below CAR activation threshold leaves the T cell silent, rendering CAR T cell therapy ineffective. With the application of CAR T cells for the treatment of a growing number of malignant diseases, particularly solid tumors, there is a need for augmenting CAR sensitivity to target antigen present at low densities on cancer cells. Here, we discuss upcoming strategies and current challenges in designing CARs for recognition of antigen low cancer cells, aiming at augmenting sensitivity and finally therapeutic efficacy while reducing the risk of tumor relapse.

Structure-function relationships of chimeric antigen receptors in acute T cell responses to antigen.

Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.

Optimization of tacrolimus in kidney transplantation: New pharmacokinetic perspectives.

Tacrolimus is the cornerstone of immunosuppressive therapy after kidney transplantation (KT), but its use is complicated by a narrow therapeutic index and high inter- and intra-patient pharmacokinetic variability. There are three available oral formulations of tacrolimus: immediate-release tacrolimus (IR-Tac), extended-release tacrolimus (ER-Tac) and a MeltDose® (LCPT) formulation, the latter favoring a prolonged drug release and increased bioavailability. The time-concentration curves of these formulations are different. Compared with IR-Tac and ER-Tac, LCPT has a relatively flat pharmacokinetic profile with less fluctuation between trough and peak exposures, and a delayed peak concentration. This translates to a more stable delivery of tacrolimus and may alleviate the risk of underexposure and allograft rejection or overexposure and toxicity. The once-daily formulation of both ER-TAC and LCPT may also offer a potential advantage on patient adherence. Fast metabolizers of tacrolimus, the elderly, and human leukocyte antigen-sensitized patients are at risk of poorer outcomes after KT, possibly associated with a different exhibited pharmacokinetics of tacrolimus or different requirements in terms of exposure. Simple, practical strategies are needed to identify patients at risk of suboptimal KT outcomes and those who would benefit from a more proactively personalized approach to tacrolimus treatment. This review aims to increase awareness of the link between the pharmacokinetics of oral tacrolimus formulations and the clinical needs of patients after KT, particularly among those who have clinically significant pharmacokinetic variation of tacrolimus.