InVivoMAb anti-human CD28
Product Details
The 9.3 monoclonal antibody reacts with human CD28, a 45 kDa costimulatory receptor and a member of the Ig superfamily. CD28 is expressed by thymocytes, most peripheral T cells, and NK cells. CD28 is a receptor for CD80 (B7-1) and CD86 (B7-2). Signaling through CD28 augments IL-2 and IL-2 receptor expression as well as cytotoxicity of CD3-activated T cells. The 9.3 antibody has been shown to stimulate the proliferation of human T cells in vitro.Specifications
| Isotype | Mouse IgG2a |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb mouse IgG2a isotype control, unknown specificity |
| Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
| Conjugation | This product is unconjugated. Conjugation is available via our Antibody Conjugation Services. |
| Immunogen | Human T lymphocytes |
| Reported Applications | in vitro T cell stimulation/activation |
| Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
| Endotoxin |
<2EU/mg (<0.002EU/Ī¼g) Determined by LAL gel clotting assay |
| Purity |
>95% Determined by SDS-PAGE |
| Sterility | 0.2 Āµm filtration |
| Production | Purified from cell culture supernatant in an animal-free facility |
| Purification | Protein G |
| RRID | AB_2687729 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze. |
Recommended Products
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Recommended Isotype Control(s)
InVivoMAb mouse IgG2a isotype control, unknown specificity
-
Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
in vitro T cell stimulation/activation
Huang, X., et al. (2021). "DNA scaffolds enable efficient and tunable functionalization of biomaterials for immune cell modulation" Nat Nanotechnol 16(2): 214-223. PubMed
Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, we developed biocompatible immune cell-engaging particles (ICEp) that use synthetic short DNA as scaffolds for efficient and tunable protein loading. To improve the safety of chimeric antigen receptor (CAR) T cell therapies, micrometre-sized ICEp were injected intratumorally to present a priming signal for systemically administered AND-gate CAR-T cells. Locally retained ICEp presenting a high density of priming antigens activated CAR T cells, driving local tumour clearance while sparing uninjected tumours in immunodeficient mice. The ratiometric control of costimulatory ligands (anti-CD3 and anti-CD28 antibodies) and the surface presentation of a cytokine (IL-2) on ICEp were shown to substantially impact human primary T cell activation phenotypes. This modular and versatile biomaterial functionalization platform can provide new opportunities for immunotherapies.
in vitro T cell stimulation/activation
Koh, J., et al. (2020). "MDSC subtypes and CD39 expression on CD8(+) T cells predict the efficacy of anti-PD-1 immunotherapy in patients with advanced NSCLC" Eur J Immunol 50(11): 1810-1819. PubMed
The major suppressive immune cells in tumor sites are myeloid derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and Treg cells, and the major roles of these suppressive immune cells include hindering T-cell activities and supporting tumor progression and survival. In this study, we analyzed the pattern of circulating MDSC subtypes in patients with non-small cell lung cancer (NSCLC) whether those suppressive immune cells hinder T-cell activities leading to poor clinical outcomes. First, we verified PMN-MDSCs, monocytic-MDSCs (M-MDSCs), and Treg cells increased according to the stages of NSCLC, and MDSCs effectively suppressed T-cell activities and induced T-cell exhaustion. The analysis of NSCLC patients treated with anti-PD-1 immunotherapy demonstrated that low PMN-MDSCs, M-MDSCs, and CD39(+) CD8(+) T cells as an individual and all together were associated with longer progression free survival and overall survival, suggesting PMN-MDSCs, M-MDSCs, and CD39(+) CD8(+) T cells frequencies in peripheral blood might be useful as potential predictive and prognostic biomarkers.
in vitro T cell stimulation/activation
Hill, E. V., et al. (2015). "Glycogen synthase kinase-3 controls IL-10 expression in CD4(+) effector T-cell subsets through epigenetic modification of the IL-10 promoter" Eur J Immunol 45(4): 1103-1115. PubMed
The serine/threonine kinase glycogen synthase kinase-3 (GSK3) plays an important role in balancing pro- and anti-inflammatory cytokines. We have examined the role of GSK3 in production of IL-10 by subsets of CD4(+) T helper cells. Treatment of naive murine CD4(+) T cells with GSK3 inhibitors did not affect their production of IL-10. However, treatment of Th1 and Th2 cells with GSK3 inhibitors dramatically increased production of IL-10. GSK3 inhibition also led to upregulation of IL-10 among Th1, Th2, and Th17 subsets isolated from human blood. The encephalitogenic potential of GSK3 inhibitor treated murine Th1 cells was significantly reduced in adoptive transfer experiments by an IL-10-dependent mechanism. Analysis of the murine IL-10 promoter in response to inhibition of GSK3 in Th1 cells showed modification to a transcriptionally active state indicated by changes in histone H3 acetylation and methylation. Additionally, GSK3 inhibition increased expression of the transcription factors c-Maf, Nfil3, and GATA3, correlating with the increase in IL-10. These findings are important in the context of autoimmune disease since they show that it is possible to reprogram disease-causing cells through GSK3 inhibition.
in vitro T cell stimulation/activation
Bushkin, Y., et al. (2015). "Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry" J Immunol 194(2): 836-841. PubMed
Flow cytometric characterization of Ag-specific T cells typically relies on detection of protein analytes. Shifting the analysis to detection of RNA would provide several significant advantages, which we illustrate by developing a new host immunity-based platform for detection of infections. Cytokine mRNAs synthesized in response to ex vivo stimulation with pathogen-specific Ags are detected in T cells with single-molecule fluorescence in situ hybridization followed by flow cytometry. Background from pre-existing in vivo analytes is lower for RNAs than for proteins, allowing greater sensitivity for detection of low-frequency cells. Moreover, mRNA analysis reveals kinetic differences in cytokine expression that are not apparent at the protein level but provide novel insights into gene expression programs expected to define different T cell subsets. The utility of probing immunological memory of infections is demonstrated by detecting T cells that recognize mycobacterial and viral Ags in donors exposed to the respective pathogens.
in vitro T cell stimulation/activation
Kovacs, B., et al. (2005). "Ligation of CD28 by its natural ligand CD86 in the absence of TCR stimulation induces lipid raft polarization in human CD4 T cells" J Immunol 175(12): 7848-7854. PubMed
Stimulation of resting CD4 T cells with anti-CD3/CD28-coated beads leads to rapid polarization of lipid rafts (LRs). It has been postulated that a major role of costimulation is to facilitate LR aggregation. CD86 is up-regulated or expressed aberrantly on immune cells in a wide array of autoimmune and infectious diseases. Using an Ig fusion with the extracellular domain of CD86 (CD86Ig) bound to a magnetic bead or K562 cells expressing CD86, we demonstrated that ligation of CD28 by its natural ligand, but not by Ab, induced polarization of LRs at the cell-bead interface of fresh human CD4 T cells in the absence of TCR ligation. This correlated with activation of Vav-1, increase of the intracellular calcium concentration, and nuclear translocation of NF-kappaB p65, but did not result in T cell proliferation or cytokine production. These studies show, for the first time, that LR polarization can occur in the absence of TCR triggering, driven solely by the CD28/CD86 interaction. This result has implications for mechanisms of T cell activation. Abnormalities in this process may alter T and B cell tolerance and susceptibility to infection.
Potent antitumor activity of a designed interleukin-21 mimic
Preprint on BioRxiv : the Preprint Server for Biology on 7 December 2024 by Chun, J., Lim, B. S., et al.
Long-standing goals of cancer immunotherapy are to activate cytotoxic antitumor T cells across a broad range of affinities while dampening suppressive regulatory T (Treg) cell responses, but current approaches achieve these goals with limited success. Here, we report a de novo IL-21 mimic, 21h10, designed to have augmented stability and high signaling potency in both humans and mice. In multiple animal models and in ex vivo human melanoma patient derived organotypic tumor spheroids (PDOTS), 21h10 showed robust antitumor activity. 21h10 generates significantly prolonged STAT signaling in vivo compared with native IL-21, and has considerably stronger anti-tumor activity. Toxicities associated with systemic administration of 21h10 could be mitigated by TNFĪ± blockade without compromising antitumor efficacy. In the tumor microenvironment, 21h10 induced highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells and driving high expression of interferon-
- Immunology and Microbiology,
Progranulin protects against Clostridioides difficile infection by enhancing IL-22 production.
In Gut Microbes on 1 October 2024 by Huang, J., Liu, B., et al.
Enhanced mortality, relapse rates, and increased prevalence of Clostridioides difficile infection (CDI) emphasize the need for better therapies and management approaches. Modulating host immune response to ameliorate CDI-associated immunopathology may provide new advantages to currently inadequate antibiotic therapies. Here, we identified progranulin (PGRN) as an important immune target upregulated in response to CDI. PGRN-deficient mice displayed dramatically higher mortality and aggravated epithelial barrier disruption compared with wild type (WT) mice after CDI despite equivalent levels of bacterial burden or toxin in the large intestine. Mechanistically, PGRN protection was mediated by IL-22 production from CD4+ T helper cells, as demonstrated by a decrease in colonic IL-22-producing CD4+ T helper cells in the intestine of PGRN-deficient mice upon CDI and a boost of IL-22-producing CD4+ T helper cells activated by PGRN ex vivo. Clinical evidence suggests that CDI patients had significantly higher serum levels of PGRN compared with healthy controls, which was significantly and positively correlated with IL-22. Our findings thus indicate a critical role for PGRN-promoted CD4+ T cell IL-22 production in shaping gut immunity and reestablishing the intestinal barrier during CDI. As an alternative to pathogen-targeted therapy, this study may provide a new host-directed therapeutic strategy to attenuate severe, refractory CDI.
- Cell Biology,
- Immunology and Microbiology
LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4+ T lymphocytes.
In Autophagy on 1 August 2024 by Pradel, B., Cantaloube, G., et al.
HIV-1 entry into CD4+ T lymphocytes relies on the viral and cellular membranes' fusion, leading to viral capsid delivery in the target cell cytoplasm. Atg8/LC3B conjugation to lipids, process named Atg8ylation mainly studied in the context of macroautophagy/autophagy, occurs transiently in the early stages of HIV-1 replication in CD4+ T lymphocytes. Despite numerous studies investigating the HIV-1-autophagy interplays, the Atg8ylation impact in these early stages of infection remains unknown. Here we found that HIV-1 exposure leads to the rapid LC3B enrichment toward the target cell plasma membrane, in close proximity with the incoming viral particles. Furthermore, we demonstrated that Atg8ylation is a key event facilitating HIV-1 entry in target CD4+ T cells. Interestingly, this effect is independent of canonical autophagy as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical step of its replication cycle.Abbreviations: BafA1: bafilomycin A1; BlaM: beta-lactamase; CD4+ TL: CD4+ T lymphocytes; PtdIns3K-BECN1 complex: BECN1-containing class III phosphatidylinositol 3-kinase complex; Env: HIV-1 envelope glycoproteins; HIV-1: type 1 human immunodeficiency virus; PM: plasma membrane; PtdIns3P: phosphatidylinositol-3-phosphate; VLP: virus-like particle.
- Cancer Research,
- Immunology and Microbiology
EGFR mutations induce the suppression of CD8+ T cell and anti-PD-1 resistance via ERK1/2-p90RSK-TGF-Ī² axis in non-small cell lung cancer.
In Journal of Translational Medicine on 14 July 2024 by Huang, H., Zhu, X., et al.
Non-small cell lung cancer (NSCLC) patients with EGFR mutations exhibit an unfavorable response to immune checkpoint inhibitor (ICI) monotherapy, and their tumor microenvironment (TME) is usually immunosuppressed. TGF-Ī² plays an important role in immunosuppression; however, the effects of TGF-Ī² on the TME and the efficacy of anti-PD-1 immunotherapy against EGFR-mutated tumors remain unclear. Corresponding in vitro studies used the TCGA database, clinical specimens, and self-constructed mouse cell lines with EGFR mutations. We utilized C57BL/6N and humanized M-NSG mouse models bearing EGFR-mutated NSCLC to investigate the effects of TGF-Ī² on the TME and the combined efficacy of TGF-Ī² blockade and anti-PD-1 therapy. The changes in immune cells were monitored by flow cytometry. The correlation between TGF-Ī² and immunotherapy outcomes of EGFR-mutated NSCLC was verified by clinical samples. We identified that TGF-Ī² was upregulated in EGFR-mutated NSCLC by EGFR activation and subsequent ERK1/2-p90RSK phosphorylation. TGF-Ī² directly inhibited CD8+ T cell infiltration, proliferation, and cytotoxicity both in vitro and in vivo, but blocking TGF-Ī² did not suppress the growth of EGFR-mutated tumors in vivo. Anti-TGF-Ī² antibody combined with anti-PD-1 antibody significantly inhibited the proliferation of recombinant EGFR-mutated tumors in C57BL/6N mice, which was superior to their monotherapy. Mechanistically, the combination of anti-TGF-Ī² and anti-PD-1 antibodies significantly increased the infiltration of CD8+ T cells and enhanced the anti-tumor function of CD8+ T cells. Moreover, we found that the expression of TGF-Ī²1 in EGFR-TKI resistant cell lines was significantly higher than that in parental cell lines. The combination of anti-TGF-Ī² and nivolumab significantly inhibited the proliferation of EGFR-TKI resistant tumors in humanized M-NSG mice and prolonged their survival. Our results reveal that TGF-Ī² expression is upregulated in NSCLC with EGFR mutations through the EGFR-ERK1/2-p90RSK signaling pathway. High TGF-Ī² expression inhibits the infiltration and anti-tumor function of CD8+ T cells, contributing to the "cold" TME of EGFR-mutated tumors. Blocking TGF-Ī² can reshape the TME and enhance the therapeutic efficacy of anti-PD-1 in EGFR-mutated tumors, which provides a potential combination immunotherapy strategy for advanced NSCLC patients with EGFR mutations. Ā© 2024. The Author(s).
- Immunology and Microbiology
Gut microbiota-derived LCA mediates the protective effect of PEDV infection in piglets.
In Microbiome on 5 February 2024 by Xing, J. H., Niu, T. M., et al.
The gut microbiota is a critical factor in the regulation of host health, but the relationship between the differential resistance of hosts to pathogens and the interaction of gut microbes is not yet clear. Herein, we investigated the potential correlation between the gut microbiota of piglets and their disease resistance using single-cell transcriptomics, 16S amplicon sequencing, metagenomics, and untargeted metabolomics. Porcine epidemic diarrhea virus (PEDV) infection leads to significant changes in the gut microbiota of piglets. Notably, Landrace pigs lose their resistance quickly after being infected with PEDV, but transplanting the fecal microbiota of Min pigs to Landrace pigs alleviated the infection status. Macrogenomic and animal protection models identified Lactobacillus reuteri and Lactobacillus amylovorus in the gut microbiota as playing an anti-infective role. Moreover, metabolomic screening of the secondary bile acids' deoxycholic acid (DCA) and lithocholic acid (LCA) correlated significantly with Lactobacillus reuteri and Lactobacillus amylovorus, but only LCA exerted a protective function in the animal model. In addition, LCA supplementation altered the distribution of intestinal T-cell populations and resulted in significantly enriched CD8+ CTLs, and in vivo and in vitro experiments showed that LCA increased SLA-I expression in porcine intestinal epithelial cells via FXR receptors, thereby recruiting CD8+ CTLs to exert antiviral effects. Overall, our findings indicate that the diversity of gut microbiota influences the development of the disease, and manipulating Lactobacillus reuteri and Lactobacillus amylovorus, as well as LCA, represents a promising strategy to improve PEDV infection in piglets. Video Abstract. Ā© 2024. The Author(s).
- Homo sapiens (Human),
- Immunology and Microbiology
Precise surface functionalization of PLGA particles for human T cell modulation.
In Nature Protocols on 1 November 2023 by Hadley, P., Chen, Y., et al.
PubMed
The biofunctionalization of synthetic materials has extensive utility for biomedical applications, but approaches to bioconjugation typically show insufficient efficiency and controllability. We recently developed an approach by building synthetic DNA scaffolds on biomaterial surfaces that enables the precise control of cargo density and ratio, thus improving the assembly and organization of functional cargos. We used this approach to show that the modulation and phenotypic adaptation of immune cells can be regulated using our precisely functionalized biomaterials. Here, we describe the three key procedures, including the fabrication of polymeric particles engrafted with short DNA scaffolds, the attachment of functional cargos with complementary DNA strands, and the surface assembly control and quantification. We also explain the critical checkpoints needed to ensure the overall quality and expected characteristics of the biological product. We provide additional experimental design considerations for modifying the approach by varying the material composition, size or cargo types. As an example, we cover the use of the protocol for human primary T cell activation and for the identification of parameters that affect ex vivo T cell manufacturing. The protocol requires users with diverse expertise ranging from synthetic materials to bioconjugation chemistry to immunology. The fabrication procedures and validation assays to design high-fidelity DNA-scaffolded biomaterials typically require 8 d. Ā© 2023. Springer Nature Limited.
- Cell Biology,
- Immunology and Microbiology
LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4+ T lymphocytes
Preprint on BioRxiv : the Preprint Server for Biology on 11 July 2023 by Pradel, B., Deffieu, M. S., et al.
PubMed
HIV-1 entry into CD4+ T lymphocytes relies on the viral and cellular membranesā fusion, leading to viral capsid delivery in the cytoplasm of target cells. The conjugation of ATG8/LC3B protein, process referred to as ATG8ylation and mainly studied in the context of autophagy, occurs transiently in the early stages of the HIV-1 replication cycle in CD4+ T lymphocytes. Despite numerous studies investigating the interplays of HIV-1 with autophagy machinery, the impact of ATG8ylation in the early stages of HIV-1 infection remains unknown. Here we found that HIV-1 exposure leads to the rapid enrichment of LC3B towards the target cell plasma membrane, in close proximity with the incoming viral particles. Furthermore, we demonstrated that ATG8ylation is a key event that facilitates HIV-1 fusion with target CD4+ T cells. Interestingly, this effect is independent of the canonical autophagy pathway as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical early step of its replication cycle. Teaser HIV-1 induces LC3B enrichment towards its target cell entry site and uses the conjugation of this protein to favor its entry step.
Selective inhibitors of JAK1 targeting an isoform-restricted allosteric cysteine.
In Nature Chemical Biology on 1 December 2022 by Kavanagh, M. E., Horning, B. D., et al.
The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity. Ā© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.
- In Vitro,
- FC/FACS,
- Homo sapiens (Human),
- Biochemistry and Molecular biology,
- Cancer Research,
- Immunology and Microbiology
WNK3 inhibition elicits antitumor immunity by suppressing PD-L1 expression on tumor cells and activating T-cell function.
In Experimental & Molecular Medicine on 1 November 2022 by Yoon, H. J., Kim, G. C., et al.
PubMed
Immune checkpoint therapies, such as programmed cell death ligand 1 (PD-L1) blockade, have shown remarkable clinical benefit in many cancers by restoring the function of exhausted T cells. Hence, the identification of novel PD-L1 regulators and the development of their inhibition strategies have significant therapeutic advantages. Here, we conducted pooled shRNA screening to identify regulators of membrane PD-L1 levels in lung cancer cells targeting druggable genes and cancer drivers. We identified WNK lysine deficient protein kinase 3 (WNK3) as a novel positive regulator of PD-L1 expression. The kinase-dead WNK3 mutant failed to elevate PD-L1 levels, indicating the involvement of its kinase domain in this function. WNK3 perturbation increased cancer cell death in cancer cell-immune cell coculture conditions and boosted the secretion of cytokines and cytolytic enzymes, promoting antitumor activities in CD4+ and CD8+ T cells. WNK463, a pan-WNK inhibitor, enhanced CD8+ T-cell-mediated antitumor activity and suppressed tumor growth as a monotherapy as well as in combination with a low-dose anti-PD-1 antibody in the MC38 syngeneic mouse model. Furthermore, we demonstrated that the c-JUN N-terminal kinase (JNK)/c-JUN pathway underlies WNK3-mediated transcriptional regulation of PD-L1. Our findings highlight that WNK3 inhibition might serve as a potential therapeutic strategy for cancer immunotherapy through its concurrent impact on cancer cells and immune cells. Ā© 2022. The Author(s).
- Homo sapiens (Human),
- COVID-19,
- Immunology and Microbiology
IL-6 drives T cell death to participate in lymphopenia in COVID-19.
In International Immunopharmacology on 1 October 2022 by Zhou, X., Ye, G., et al.
PubMed
Lymphopenia is a common observation in patients with COVID-19. To explore the cause of T cell lymphopenia in the disease, laboratory results of 64 hospitalized COVID-19 patients were retrospectively analyzed and six patients were randomly selected to trace their changes of T lymphocytes and plasma concentration of IL-6 for the course of disease. Results confirmed that the T-cell lymphopenia, especially CD4+ T cell reduction in COVID-19 patients, was a reliable indicator of severity and hospitalization in infected patients. And CD4+ T cell count below 200 cells/Ī¼L predicts critical illness in COVID-19 patients. In vitro assay supported that exposure to key contributors (IL-1Ī², IL-6, TNF-Ī± and IFN-Ī³) of COVID-19 cytokine storm caused substantial death of activated T cells. Among these contributors, IL-6 level was found to probably reversely correlate with T cell counts in patients. And IL-6 alone was potent to induce T cell reduction by gasderminE-mediated pyroptosis, inferring IL-6 took a part in affecting the function and status of T cells in COVID-19 patients. Intervention of IL-6 mediated T cell pryprotosis may effectively delay disease progression, maintain normal immune status at an early stage of infection. Copyright Ā© 2022 Elsevier B.V. All rights reserved.
- Control,
- Homo sapiens (Human),
- Biochemistry and Molecular biology
Discovery of Aptamers Against Cell Surface Markers Using Ligand-Guided Selection.
In Methods in Molecular Biology (Clifton, N.J.) on 27 September 2022 by Williams, N., Patel, R., et al.
PubMed
Oligonucleotide ligands (DNA, RNA, or XNA), also known as aptamers, are selected against various target molecules using an iterative, evolutionary process called systematic evolution of ligands by exponential enrichment (SELEX). To select aptamers against complex cell surface proteins in their native state, a variant of SELEX termed ligand-guided selection (LIGS) was recently introduced. The significance of LIGS is rooted in its strategy of exploiting the selection step in SELEX to identify highly specific aptamers against known cell surface markers. Thus, in LIGS, a higher-affinity secondary ligand, such as a monoclonal antibody (mAb) to a whole-cell bound to an evolved SELEX library, is introduced to outcompete sequences against the mAb targeting cell surface protein or induce a conformational switch to destabilize the aptamer-surface cell surface protein resulting in elution of the sequences. Here, we describe the detailed method of LIGS utilized in identifying aptamers against T-cell receptor cluster of differentiation three complex (TCR-CD3) expressed in human T-cells and T-cell leukemia. Ā© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
- Cancer Research,
- Immunology and Microbiology
Balance between immunoregulatory B cells and plasma cells drives pancreatic tumor immunity.
In Cell Reports Medicine on 20 September 2022 by Mirlekar, B., Wang, Y., et al.
PubMed
Plasma cell responses are associated with anti-tumor immunity and favorable response to immunotherapy. BĀ cells can amplify anti-tumor immune responses through antibody production; yet B cells in patients and tumor-bearing mice often fail to support this effector function. We identify dysregulated transcriptional program in B cells that disrupts differentiation of naive B cells into anti-tumor plasma cells. The signaling network contributing to this dysfunction is driven by interleukin (IL) 35 stimulation of a STAT3-PAX5 complex that upregulates the transcriptional regulator BCL6 in naive B cells. Transient inhibition of BCL6 in tumor-educated naive B cells is sufficient to reverse the dysfunction in B cell differentiation, stimulating the intra-tumoral accumulation of plasma cells and effector TĀ cells and rendering pancreatic tumors sensitive to anti-programmed cell death protein 1 (PD-1) blockade. Our findings argue that B cell effector dysfunction in cancer can be due to an active systemic suppression program that can be targeted to synergize with TĀ cell-directed immunotherapy. Copyright Ā© 2022 The Authors. Published by Elsevier Inc. All rights reserved.
- Cancer Research,
- Immunology and Microbiology
Intracellular Acetyl CoA Potentiates the Therapeutic Efficacy of Antitumor CD8+ T Cells.
In Cancer Research on 18 July 2022 by Chowdhury, S., Kar, A., et al.
PubMed
Effector CD8+ T cells rely primarily on glucose metabolism to meet their biosynthetic and functional needs. However, nutritional limitations in the tumor microenvironment can cause T-cell hyporesponsiveness. Therefore, T cells must acquire metabolic traits enabling sustained effector function at the tumor site to elicit a robust antitumor immune response. Here, we report that IL12-stimulated CD8+ T cells have elevated intracellular acetyl CoA levels and can maintain IFNĪ³ levels in nutrient-deprived, tumor-conditioned media (TCM). Pharmacological and metabolic analyses demonstrated an active glucose-citrate-acetyl CoA circuit in IL12-stimulated CD8+ T cells supporting an intracellular pool of acetyl CoA in an ATP-citrate lyase (ACLY)-dependent manner. Intracellular acetyl CoA levels enhanced histone acetylation, lipid synthesis, and IFNĪ³ production, improving the metabolic and functional fitness of CD8+ T cells in tumors. Pharmacological inhibition or genetic knockdown of ACLY severely impaired IFNĪ³ production and viability of CD8+ T cells in nutrient-restricted conditions. Furthermore, CD8+ T cells cultured in high pyruvate-containing media in vitro acquired critical metabolic features of IL12-stimulated CD8+ T cells and displayed improved antitumor potential upon adoptive transfer in murine lymphoma and melanoma models. Overall, this study delineates the metabolic configuration of CD8+ T cells required for stable effector function in tumors and presents an affordable approach to promote the efficacy of CD8+ T cells for adoptive T-cell therapy. IL12-mediated metabolic reprogramming increases intracellular acetyl CoA to promote the effector function of CD8+ T cells in nutrient-depleted tumor microenvironments, revealing strategies to potentiate the antitumor efficacy of T cells. Ā©2022 The Authors; Published by the American Association for Cancer Research.
- Cell Culture,
- Homo sapiens (Human)
Ursodeoxycholic acid reduces antitumor immunosuppression by inducing CHIP-mediated TGF-Ī² degradation.
In Nature Communications on 14 June 2022 by Shen, Y., Lu, C., et al.
PubMed
TGF-Ī² is essential for inducing systemic tumor immunosuppression; thus, blocking TGF-Ī² can greatly enhance antitumor immunity. However, there are still no effective TGF-Ī² inhibitors in clinical use. Here, we show that the clinically approved compound ursodeoxycholic acid (UDCA), by degrading TGF-Ī², enhances antitumor immunity through restraining Treg cell differentiation and activation in tumor-bearing mice. Furthermore, UDCA synergizes with anti-PD-1 to enhance antitumor immunity and tumor-specific immune memory in tumor-bearing mice. UDCA phosphorylates TGF-Ī² at T282 site via TGR5-cAMP-PKA axis, causing increased binding of TGF-Ī² to carboxyl terminus of Hsc70-interacting protein (CHIP). Then, CHIP ubiquitinates TGF-Ī² at the K315 site, initiating p62-dependent autophagic sorting and subsequent degradation of TGF-Ī². Notably, results of retrospective analysis shows that combination therapy with anti-PD-1 or anti-PD-L1 and UDCA has better efficacy in tumor patients than anti-PD-1 or anti-PD-L1 alone. Thus, our results show a mechanism for TGF-Ī² regulation and implicate UDCA as a potential TGF-Ī² inhibitor to enhance antitumor immunity. Ā© 2022. The Author(s).
- Cell Culture,
- Homo sapiens (Human),
- COVID-19,
- Immunology and Microbiology
Effective chimeric antigen receptor T cells against SARS-CoV-2.
In IScience on 19 November 2021 by Guo, X., Kazanova, A., et al.
PubMed
Current therapies to treat coronavirus disease 2019 (COVID-19) involve vaccines against the spike protein S1 of SARS-CoV-2. Here, we outline an alternative approach involving chimeric antigen receptors (CARs) in TĀ cells (CAR-Ts). CAR-T recognition of the SARS-CoV-2 receptor-binding domain (RBD) peptide induced ribosomal protein S6 phosphorylation, the increased expression of activation antigen, CD69 and effectors, interferon-Ī³, granzyme B, perforin, and Fas-ligand on overlapping subsets of CAR-Ts. CAR-Ts further showed potent inĀ vitro killing of target cells loaded with RBD, S1 peptide, or expressing the S1 protein. The efficacy of killing varied with different sized hinge regions, whereas time-lapse microscopyĀ showed CAR-T cluster formation around RBD-expressing targets. Cytolysis of targets was mediated primarily by the GZMB/perforin pathway. Lastly, we showed inĀ vivo killing of S1-expressing cells by our SARS-CoV-2 CAR-Ts in mice. The successful generation of SARS-CoV-2 CAR-Ts represents a living vaccine approach for the treatment of COVID-19. Crown Copyright Ā© 2021.
- Cell Culture,
- Homo sapiens (Human),
- Immunology and Microbiology
NKD2 mediates stimulation-dependent ORAI1 trafficking to augment Ca2+ entry in TĀ cells.
In Cell Reports on 24 August 2021 by Wu, B., Woo, J. S., et al.
PubMed
Sustained activation of the Ca2+-release-activated Ca2+ (CRAC) channel is pivotal for effector TĀ cell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector TĀ cells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we identify an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of TĀ cell receptors, orchestrates trafficking and insertion of ORAI1+ vesicles to the plasma membrane. Together, our findings suggest that TĀ cell receptor (TCR)-stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in TĀ cells. Copyright Ā© 2021 The Author(s). Published by Elsevier Inc. All rights reserved.
- In Vitro,
- Homo sapiens (Human),
- Immunology and Microbiology
Multiplexed proteomic profiling of cysteine reactivity and ligandability in human TĀ cells.
In STAR Protocols on 18 June 2021 by Vinogradova, E. V. & Cravatt, B. F.
PubMed
Differential amino acid reactivity with chemical probes can provide valuable information on the functionality and ligandability of proteins in native biological systems. Here, we present a quantitative, multiplexed chemical proteomic protocol for in-depth reactivity and ligandability profiling of cysteines in proteins in quiescent and stimulated TĀ cells. This protocol illuminates dynamic immune state-dependent alterations in cysteine reactivity, revealing chemoselective and stereoselective small-molecule interactions with cysteines in structurally and functionally diverse proteins that lack chemical probes. For complete details on the use and execution of this protocol, please refer to Vinogradova etĀ al. (2020). Ā© 2021 The Authors.
- Binding,
- Homo sapiens (Human),
- Immunology and Microbiology
Biphasic response of T cell activation to substrate stiffness.
In Biomaterials on 1 June 2021 by Yuan, D. J., Shi, L., et al.
PubMed
T cell activation is sensitive to the mechanical properties of an activating substrate. However, there are also contrasting results on how substrate stiffness affects T cell activation, including differences between T cells of mouse and human origin. Towards reconciling these differences, this report examines the response of primary human T cells to polyacrylamide gels with stiffness between 5 and 110Ā kPa presenting activating antibodies to CD3 and CD28. T cell proliferation and IL-2 secretion exhibited a biphasic functional response to substrate stiffness, which can be shifted by changing density of activating antibodies and abrogated by inhibition of cellular contractility. T cell morphology was modulated by stiffness at early time points. RNA-seq indicates that T cells show differing monotonic trends in upregulated genes and pathways towards both ends of the stiffness spectrum. These studies provide a framework of T cell mechanosensing and suggest an effect of ligand density that may reconcile different, contrasting patterns of stiffness sensing seen in previous studies. Copyright Ā© 2021 Elsevier Ltd. All rights reserved.
- Immunology and Microbiology
Semiflexible Immunobrushes Induce Enhanced T Cell Activation and Expansion.
In ACS Applied Materials Interfaces on 14 April 2021 by Hammink, R., Weiden, J., et al.
PubMed
A variety of bioactive materials developed to expand T cells for adoptive transfer into cancer patients are currently evaluated in the clinic. In most cases, T cell activating biomolecules are attached to rigid surfaces or matrices and form a static interface between materials and the signaling receptors on the T cells. We hypothesized that a T cell activating polymer brush interface might better mimic the cell surface of a natural antigen-presenting cell, facilitating receptor movement and concomitant advantageous mechanical forces to provide enhanced T cell activating capacities. Here, as a proof of concept, we synthesized semiflexible polyisocyanopeptide (PIC) polymer-based immunobrushes equipped with T cell activating agonistic anti-CD3 (Ī±CD3) and Ī±CD28 antibodies placed on magnetic microbeads. We demonstrated enhanced efficiency of ex vivo expansion of activated primary human T cells even at very low numbers of stimulating antibodies compared to rigid beads. Importantly, the immunobrush architecture appeared crucial for this improved T cell activating capacity. Immunobrushes outperform current benchmarks by producing higher numbers of T cells exhibiting a combination of beneficial phenotypic characteristics, such as reduced exhaustion marker expression, high cytokine production, and robust expression of cytotoxic hallmarks. This study indicates that semiflexible immunobrushes have great potential in making T cell-based immunotherapies more effective.
KIR3DL3 Is an Inhibitory Receptor for HHLA2 that Mediates an Alternative Immunoinhibitory Pathway to PD1.
In Cancer Immunology Research on 1 February 2021 by Bhatt, R. S., Berjis, A., et al.
PubMed
Blockade of the PD1 pathway is a broadly effective cancer therapy, but additional immune-inhibitory pathways contribute to tumor immune evasion. HERV-H LTR-associating 2 (HHLA2; also known as B7H5 and B7H7) is a member of the B7 family of immunoregulatory ligands that mediates costimulatory effects through its interaction with the CD28 family member transmembrane and immunoglobulin domain containing 2 (TMIGD2). However, HHLA2 has also been known to have inhibitory effects on T cells. Here, we report that we have identified killer cell immunoglobulin-like receptor, three immunoglobulin domains and long cytoplasmic tail 3 (KIR3DL3) as an inhibitory receptor for HHLA2 in T cells and natural killer (NK) cells and have generated HHLA2 and KIR3DL3 antibodies that block the immune-inhibitory activity of HHLA2, preserving the costimulatory signal. It is known that HHLA2 is frequently expressed in several tumor types, including clear cell renal cell carcinoma (ccRCC). We found that HHLA2 expression was nonoverlapping with PDL1 expression in ccRCC, suggesting that HHLA2 mediates a mechanism of tumor immune evasion that is independent from PDL1. Blockade of both the PD1 and KIR3DL3 pathways may be a more effective way to reverse tumor immune evasion.See related Spotlight on p. 128. Ā©2020 American Association for Cancer Research.
