Phospholipases constitute a ubiquitous class of membrane-active enzymes that play a key role in cellular signaling, proliferation, and membrane trafficking. Aberrant phospholipase activity is ...implicated in a range of diseases including cancer, inflammation, and myocardial disease. Characterization of these enzymes is therefore important, both for improving the understanding of phospholipase catalysis and for accelerating pharmaceutical and biotechnological applications. This paper describes a novel approach to monitor, in situ and in real-time, the activity of phospholipase D (PLD) and phospholipase C (PLC) on planar lipid bilayers. This method is based on lipase-induced changes in the electrical charge of lipid bilayers and on the concomitant change in ion concentration near lipid membranes. The approach reports these changes in local ion concentration by a measurable change in the single channel ion conductance through pores of the ion channel-forming peptide gramicidin A. This enzyme assay takes advantage of the amplification characteristics of gramicidin pores to sense the activity of picomolar to nanomolar concentrations of membrane-active enzymes without requiring labeled substrates or products. The resulting method proceeds on lipid bilayers without the need for detergents, quantifies enzyme activity on native lipid substrates within minutes, and provides unique access to both leaflets of well-defined lipid bilayers; this method also makes it possible to generate planar lipid bilayers with transverse lipid asymmetry.
Unlike biological protein pores in lipid membranes, nanopores fabricated in synthetic materials can withstand a wide range of environmental conditions including the presence of organic solvents. This ...capability expands the potential of synthetic nanopores to monitor chemical reactions occurring at the interface between solutions of organic and aqueous character. In this work, nanopores fabricated in borosilicate glass or silicon nitride connected a predominantly organic solvent to an aqueous solvent, thereby generating a mixing zone between these solutions inside the pore. This configuration was exploited to precipitate small organic molecules with low aqueous solubility inside the nanopores, and concomitantly, to monitor this precipitation by the decrease of the ionic conductance through the nanopores over time. Hence, this method provides a means to induce and investigate the formation of nanoprecipitates or nanoparticles. Interestingly, precipitates with a slight electric charge were cleared from the pore, causing the conductance of the pore to return to its original value. This process repeated, resulting in stable oscillations of the ionic current. Although such oscillations might be useful in fluidic logic circuits, few conditions capable of generating oscillations in ionic currents have been reported. The frequency and amplitude of oscillations could be tuned by changing the concentration of the precipitating molecule, the pH of the electrolyte, and the applied potential bias. In addition to generating oscillations, nanopores that separate two different solutions may be useful for monitoring and mediating chemical reactions in the mixing zone between two solutions.
T cell alloreactivity is mediated by a self-human leukocyte antigen (HLA)-restricted T cell receptor (TCR) repertoire able to recognize both structurally similar and dissimilar allogeneic HLA ...molecules (i.e., differing by a single or several amino acids in their peptide-binding groove). We hypothesized that thymic selection on self-HLA molecules could have an indirect impact on the size and diversity of the alloreactive response. To test this possibility, we used TCR Vβ immunophenotyping and immunosequencing technology in a model of alloreactivity between self-HLA selected T cells and allogeneic HLA-DPB1 (DPB1) differing from self-DPB1*04:02 by a single (DPB1*02:01) or several (DPB1*09:01) amino acids in the peptide-binding groove. CD4+ T cells from three different self-DPB1*04:01,*04:02 individuals were stimulated with HeLa cells stably transduced with the relevant peptide processing machinery, co-stimulatory molecules, and HLA-DP. Flow cytometric quantification of the DPB1-specific T cell response measured as upregulation of the activation marker CD137 revealed significantly lower levels of alloreactivity against DPB1*02:01 compared with DPB1*09:01 (mean CD4+CD137+ frequency 35.2 ± 9.9 vs. 61.5 ± 7.7%, respectively,
< 0.0001). These quantitative differences were, however, not reflected by differences in the breadth of the alloreactive response at the Vβ level, with both alloantigens eliciting specific responses from all TCR-Vβ specificities tested by flow cytometry, albeit with higher levels of reactivity from most Vβ specificities against DPB1*09:01. In line with these observations,
-CDR3 immunosequencing showed no significant differences in mean clonality of sorted CD137+CD4+ cells alloreactive against DPB1*02:01 or DPB1*09:01 0.39 (0.36-0.45) and 0.39 (0.30-0.46), respectively, or in the cumulative frequencies of the 10 most frequent responding clones (55-67 and 58-62%, respectively). Most of the clones alloreactive against DPB1*02:01 (68.3%) or DPB1*09:01 (75.3%) were characterized by low-abundance (i.e., they were not appreciable among the pre-culture T cells). Interestingly, however, their cumulative frequency was lower against DPB1*02:01 compared with DPB1*09:01 (mean cumulative frequency 35.3 vs. 50.6%, respectively). Our data show that, despite lower levels of alloreactivity, a similar clonal diversity can be elicited by structurally similar compared with structurally dissimilar HLA-DPB1 alloantigens and demonstrate the power of
immunosequencing in unraveling subtle qualitative changes not appreciable by conventional methods.
We demonstrate rapid fabrication of submicrometer-diameter pores in borosilicate glass using femtosecond laser machining and subsequent wet-etch techniques. This approach allows direct and repeatable ...fabrication of high-quality pores with diameters of 400-800 nm. Such small pores coupled with the desirable electrical and chemical properties of glass enable sensitive resistive-pulse analysis to determine the size and concentration of macromolecules and nanoparticles. Plasma-enhanced chemical vapor deposition allows further reduction of pore diameters to below 300 nm.
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Background: Patients with metastatic melanoma were treated on a clinical trial with tremelimumab and High Dose Interferon-Alfa (HDI) (Tarhini. J Clin Oncol. 2012). We previously ...reported that patients who achieved disease control and clinical response had significantly greater T-cell clonality (p = 0.0008) and T-cell fraction (p = 0.044) respectively in their pretreatment tumor biopsy samples (Tarhini. J Clin Oncol. 2017). In this study, we further characterize T-cell repertoire clonality and clonal expansion in the peripheral blood at different time points to evaluate the association between repertoire features and clinical response. Methods: Patients received tremelimumab 15 mg/kg I.V. every 12 weeks and HDI was given concurrently. Responses were assessed by RECIST as complete (CR) or partial (PR), stable disease (SD) or progression (PD). Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 33) were obtained at baseline, day 29, and day 85 (following tremelimumab-HDI treatment); tumor samples at baseline were also obtained (N = 18). The T-cell receptor beta chain (TCRB) repertoire of PBMCs and tumor samples was immunosequenced using the immunoSEQ assay (Adaptive Biotechnologies), and repertoire clonality was assessed at baseline, day 29, and day 85. Differential abundance analysis was used to detect and quantify peripheral clonal expansion pre- versus post-treatment and identify the subset of peripheral clones also detected in the tumor repertoire. The Morisita Index of repertoire similarity was also calculated to compare global repertoire changes between pre- and post-treatment PBMC samples. Results: T-cell repertoire turnover, as measured by the Morisita Index, showed a trend towards responders (CR/PR) having greater turnover (lower Morisita Index) post-treatment than non-responders (SD/PD). Similarly, the total number of clones expanding in the peripheral repertoire varied over time within an individual (p = 0.034) but was not significantly affected by response to therapy (p = 0.275) or by on-treatment time point (p = 0.768). When the analysis was restricted to peripherally expanded clones that were also found in the tumor repertoire, responders had significantly more TILs expanded in the periphery at day 29 than non-responders (p = 0.036). Conclusions: Our analysis of the peripheral T-cell repertoire following treatment showed that detection of TILs in early peripheral clonal expansion correlates with response to therapy.
Immune checkpoint therapies, such as ipilimumab, induce dramatic antitumor responses in a subset of patients with advanced malignancies, but they may also induce inflammatory responses and toxicities ...termed immune-related adverse events (irAEs). These irAEs are often low grade and manageable, but severe irAEs may lead to prolonged hospitalizations or fatalities. Early intervention is necessary to minimize morbidities that occur with severe irAEs. However, correlative biomarkers are currently lacking. In a phase II clinical trial that treated 27 patients with metastatic prostate cancer, we aimed to test the safety and efficacy of androgen deprivation therapy plus ipilimumab. In this study, we observed grade 3 toxicities in >40% of treated patients, which led to early closure of the study. Because ipilimumab enhances T-cell responses, we hypothesized that increased clonal T-cell responses in the systemic circulation may contribute to irAEs. Sequencing of the T-cell receptor β-chains in purified T cells revealed clonal expansion of CD8 T cells, which occurred in blood samples collected before the onset of grade 2–3 irAEs. These initial results suggested that expansion of ≥55 CD8 T-cell clones preceded the development of severe irAEs. We further evaluated available blood samples from a second trial and determined that patients who experienced grade 2–3 irAEs also had expansion of ≥55 CD8 T-cell clones in blood samples collected before the onset of irAEs. We propose that CD8 T-cell clonal expansion may be a correlative biomarker to enable close monitoring and early intervention for patients receiving ipilimumab.
Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of ...patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy.
Abstract
Delayed reconstitution of the immune system after allogeneic hematopoietic cell transplantation (HCT) is a long-recognized complication. Specifically, loss of T-cell diversity has been ...implicated in infectious complications, graft-versus-host disease (GVHD), and disease relapse. We performed serial high-resolution next generation sequencing using the immunoSEQ® Assay (Adaptive Biotechnologies, Seattle, WA) to characterize the TCRβ locus in 99 HCT recipients in the first 3 months after transplant. Transplant donor type included unrelated (n=57) and related (n=42) donors. Conditioning regimen intensity included reduced intensity (n=55) and myeloablative (n=44). We measured T-cell fraction, clonality, and richness in the donor and at days +15, +30, +50 and +100 post-transplant in the recipient, and correlated metrics to clinical variables. In agreement with prior studies, we found that although absolute T-cell numbers recover relatively quickly after transplant, repertoire diversity remains diminished. Restricted diversity was associated with conditioning intensity, use of ATG, and donor type. Increased T-cell clonal expansion at Day +30 compared to the donor sample was associated with the incidence of acute GVHD (HR=1.11, p=5x10-5). Even after exclusion of the twelve patients who had experienced acute GVHD by day +30, the association between acute GVHD and clonal expansion persisted (HR=1.098, p=0.041), indicating that clonal expansion preceded the development of acute GVHD. Our results indicate the importance of early post-transplant sampling and highlight T-cell clonal expansion as a potential novel biomarker for GVHD which warrants further study.
Citation Format: Rachel M. Gittelman, Mark Leick, Zachariah DeFilipp, Jerome Ritz, Erik Yusko, Catherine Sanders, Harlan Robins. T-cell clonal dynamics determined by high resolution TCR-β sequencing in recipients of allogeneic hematopoietic cell transplantation abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-272.
The Bruton's tyrosine kinase inhibitor ibrutinib is a highly effective, new targeted therapy for chronic lymphocytic leukemia (CLL) that thwarts leukemia cell survival, growth, and tissue homing. The ...effects of ibrutinib treatment on the T cell compartment, which is clonally expanded and thought to support the growth of malignant B cells in CLL, are not fully characterized. Using next-generation sequencing technology, we characterized the diversity of TCRβ-chains in peripheral blood T cells from 15 CLL patients before and after 1 y of ibrutinib therapy. We noted elevated CD4
and CD8
T cell numbers and a restricted TCRβ repertoire in all pretreatment samples. After 1 y of ibrutinib therapy, elevated peripheral blood T cell numbers and T cell-related cytokine levels had normalized, and T cell repertoire diversity increased significantly. Dominant TCRβ clones in pretreatment samples declined or became undetectable, and the number of productive unique clones increased significantly during ibrutinib therapy, with the emergence of large numbers of low-frequency TCRβ clones. Importantly, broader TCR repertoire diversity was associated with clinical efficacy and lower rates of infections during ibrutinib therapy. These data demonstrate that ibrutinib therapy increases diversification of the T cell compartment in CLL patients, which contributes to cellular immune reconstitution.
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