scholarly journals Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells

2018 ◽  
Vol 215 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Akiko Seki ◽  
Sascha Rutz
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Editing ◽  
Target Gene ◽  
Gene Knockout ◽  
Great Promise ◽  
Next Generation ◽  
Primary Mouse

CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.

NFATc3 regulates the transcription of genes involved in T-cell activation and angiogenesis

Blood ◽  
2011 ◽  
Vol 118 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Katia Urso ◽  
Arantzazu Alfranca ◽  
Sara Martínez-Martínez ◽  
Amelia Escolano ◽  
Inmaculada Ortega ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Gene Knockout ◽  
Activated T Cells ◽  
Knock Down ◽  
And Function

Abstract The nuclear factor of activated T cells (NFAT) family of transcription factors plays important roles in many biologic processes, including the development and function of the immune and vascular systems. Cells usually express more than one NFAT member, raising the question of whether NFATs play overlapping roles or if each member has selective functions. Using mRNA knock-down, we show that NFATc3 is specifically required for IL2 and cyclooxygenase-2 (COX2) gene expression in transformed and primary T cells and for T-cell proliferation. We also show that NFATc3 regulates COX2 in endothelial cells, where it is required for COX2, dependent migration and angiogenesis in vivo. These results indicate that individual NFAT members mediate specific functions through the differential regulation of the transcription of target genes. These effects, observed on short-term suppression by mRNA knock-down, are likely to have been masked by compensatory effects in gene-knockout studies.

scholarly journals ROG Negatively Regulates T-Cell Activation but Is Dispensable for Th-Cell Differentiation

2005 ◽  
Vol 25 (2) ◽  
pp. 554-562 ◽  
Author(s):  
Bok Yun Kang ◽  
Shi-Chuen Miaw ◽  
I-Cheng Ho
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Gene ◽  
Interleukin 2 ◽  
Negative Regulator ◽  
Th2 Cells ◽  
Th Cells ◽  
And Function

ABSTRACT ROG, a transcriptional repressor, is a direct target gene of NF-AT and a putative negative regulator of T-cell activation. In addition, overexpression of ROG suppresses the activity of GATA-3, implying a role of ROG in the differentiation and function of Th cells. Despite these observations, the function of ROG has yet to be confirmed by loss-of-function approaches. Here we report that ROG-deficient T cells are hypersensitive to anti-CD3 stimulation and produce more interleukin-2 (IL-2) due to enhanced NF-κB activity. ROG-deficient dendritic cells also produce more IL-12p40, another NF-κB target gene. However, ROG-deficient Th cells are capable of differentiating into Th1 and Th2 cells, and ROG-deficient mice have no defect in mounting appropriate Th immune responses in vivo. Thus, ROG is dispensable for the differentiation and function of Th cells but serves as a mediator of NF-AT-initiated suppression of NF-κB. Its mechanism of action and its expression pattern are distinct from those of other transcription factors negatively regulating the activation of T cells.

scholarly journals Induction of Antigen-Specific T-Cell Responses through Dendritic Cell Vaccination in AML: Results of a Phase I/II Trial and Ex Vivo Enhancement By Checkpoint Blockade

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 764-764 ◽  
Author(s):  
Felix S. Lichtenegger ◽  
Katrin Deiser ◽  
Maurine Rothe ◽  
Frauke M. Schnorfeil ◽  
Christina Krupka ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Phase I ◽  
T Cell Activation ◽  
Cell Activation ◽  
Next Generation ◽  
Cell Responses ◽  
Ifn Γ

Abstract Postremission therapy is critical for successful elimination of minimal residual disease (MRD) in acute myeloid leukemia (AML). Innovative treatment options are needed for patients that are not eligible for allogeneic stem cell transplantation. As the intrinsic immune response against leukemia-associated antigens (LAAs) is generally low, the clinical application of checkpoint inhibitors as monotherapy is less promising in AML compared to other hemato-oncological diseases. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with LAAs is a promising treatment strategy to induce anti-leukemic immune responses. We have conducted a phase I/II proof-of-concept study using monocyte-derived next-generation DCs as postremission therapy of AML patients with a non-favorable risk profile in CR/CRi after intensive induction therapy (NCT01734304). These DCs are generated using a GMP-compliant 3-day protocol including a TLR7/8 agonist, loaded with RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant/surrogate antigen, and are applied intradermally up to 10 times within 26 weeks. The primary endpoint of the trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. After the safety and toxicity profile was evaluated within phase I (n=6), the patient cohort was expanded to a total of 13 patients. DCs of sufficient number and quality could be generated from leukapheresis in 11/12 cases. DCs exhibited an immune-stimulatory profile based on high costimulatory molecule expression, IL-12p70 secretion, migration towards a chemokine gradient and processing and presentation of antigen. In 9/9 patients that are currently evaluable, we observed delayed-type hypersensitivity (DTH) responses at the vaccination site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. In the peripheral blood, we detected vaccination-specific T cell responses by multimer staining and by ELISPOT analysis: 7/7 patients showed responses to CMVpp65, including both boosting of pre-existing T cells in CMV+ patients and induction of a primary T cell response in CMV- patients. 2/7 patients exhibited responses to PRAME and WT each. 7/10 vaccinated patients are still alive, and 5/10 are in CR, with an observation period of up to 840 days. In vitro, DC-activated T cells showed an upregulation of PD-1 and LAG-3, while the DCs expressed the respective ligands PD-L1 and HLA-DR. Therefore, we studied the capacity of checkpoint blocking antibodies to further enhance T-cell activation by DCs. We found that blockade of PD-1 and particularly of LAG-3 was highly effective in enhancing both IFN-γ secretion and proliferation of T cells. Both pathways seem to target different T-cell subsets, as PD-1 blockade resulted in increased IFN-γ secretion by TN- and TEM-subsets, while blockade of LAG-3 significantly affected TN- and TCM-subsets. We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe, and induces anti-leukemic immune responses in vivo. Our in vitro data supports the hypothesis that T-cell activation by means of the vaccine could be further enhanced by blocking PD-1 and/or LAG-3. Disclosures Subklewe: AMGEN Research Munich: Research Funding.

scholarly journals CD137 as an Attractive T Cell Co-Stimulatory Target in the TNFRSF for Immuno-Oncology Drug Development

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2288
Author(s):  
Kenji Hashimoto
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
T Cell ◽  
Clinical Efficacy ◽  
T Cell Activation ◽  
Cell Activation ◽  
Checkpoint Inhibitors ◽  
Antibody Engineering ◽  
Cytotoxic T Cell ◽  
Next Generation

Immune checkpoint inhibitors have altered the treatment landscape significantly in several cancers, yet not enough for many cancer patients. T cell costimulatory receptors have been pursued as targets for the next generation of cancer immunotherapies, however, sufficient clinical efficacy has not yet been achieved. CD137 (TNFRSF9, 4-1BB) provides co-stimulatory signals and activates cytotoxic effects of CD8+ T cells and helps to form memory T cells. In addition, CD137 signalling can activate NK cells and dendritic cells which further supports cytotoxic T cell activation. An agonistic monoclonal antibody to CD137, urelumab, provided promising clinical efficacy signals but the responses were achieved above the maximum tolerated dose. Utomilumab is another CD137 monoclonal antibody to CD137 but is not as potent as urelumab. Recent advances in antibody engineering technologies have enabled mitigation of the hepato-toxicity that hampered clinical application of urelumab and have enabled to maintain similar potency to urelumab. Next generation CD137 targeting molecules currently in clinical trials support T cell and NK cell expansion in patient samples. CD137 targeting molecules in combination with checkpoint inhibitors or ADCC-enhancing monoclonal antibodies have been sought to improve both clinical safety and efficacy. Further investigation on patient samples will be required to provide insights to understand compensating pathways for future combination strategies involving CD137 targeting agents to optimize and maintain the T cell activation status in tumors.

scholarly journals Macropinocytosis drives T cell growth by sustaining the activation of mTORC1

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
John C. Charpentier ◽  
Di Chen ◽  
Philip E. Lapinski ◽  
Jackson Turner ◽  
Irina Grigorova ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Growth ◽  
T Cell Activation ◽  
Mammalian Cells ◽  
Large Scale ◽  
Cell Activation ◽  
Phase Form ◽  
Primary Mouse ◽  
T Cell Growth

AbstractMacropinocytosis is an evolutionarily-conserved, large-scale, fluid-phase form of endocytosis that has been ascribed different functions including antigen presentation in macrophages and dendritic cells, regulation of receptor density in neurons, and regulation of tumor growth under nutrient-limiting conditions. However, whether macropinocytosis regulates the expansion of non-transformed mammalian cells is unknown. Here we show that primary mouse and human T cells engage in macropinocytosis that increases in magnitude upon T cell activation to support T cell growth even under amino acid (AA) replete conditions. Mechanistically, macropinocytosis in T cells provides access of extracellular AA to an endolysosomal compartment to sustain activation of the mechanistic target of rapamycin complex 1 (mTORC1) that promotes T cell growth. Our results thus implicate a function of macropinocytosis in mammalian cell growth beyond Ras-transformed tumor cells via sustained mTORC1 activation.

scholarly journals Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples

2021 ◽  
Author(s):  
Anneke Eerkens ◽  
Annege Vledder ◽  
Nienke van Rooij ◽  
Floris Foijer ◽  
Hans W Nijman ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Editing ◽  
Ex Vivo ◽  
Immune Monitoring ◽  
Functional Evaluation ◽  
Starting Point

Objectives: CRISPR/Cas9-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during e.g. clinical trials. Methods: PBMCs from healthy donors were used to generate knockout T cell models for interferon-gamma (IFNg), Cbl Proto-Oncogene B (CBLB), Fas cell surface death receptor (Fas) and T cell receptor (TCR alpha and beta) genes. The effect of CRISPR-cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays. Results: Our results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T cell viability. Cytokine release and T cell proliferation were not affected in gene edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naive T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T cell responses in gene-edited and untouched control cells; making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T cell activation and expansion assays. Conclusion: Here, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically-used immune-monitoring pipelines and serves as a starting point for development of analogous approaches such as those including transcriptional activators and or epigenetic modifiers.

TCR Gene Editing Achieved In a Single Round Of T Cell Activation Is Sufficient To Redirect T Cell Specificity and Prevent GvHD

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2898-2898
Author(s):  
Sara Mastaglio ◽  
Pietro Genovese ◽  
Zulma Magnani ◽  
Elena Provasi ◽  
Angelo Lombardo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
T Cell Activation ◽  
Cell Activation ◽  
Gene Editing ◽  
Hematological Malignancies ◽  
Myeloma Cell ◽  
Hematopoietic Stem ◽  
Cell Specificity

Abstract The genetic transfer of tumor-specific T cell receptors (TCRs) into mature T lymphocytes enables T cell specificity to be redirected towards cancer cells, however the transfer of novel TCRs into polyclonal T cells, while overcoming tolerance barriers, may be limited by factors intrinsic to TCR biology. Specifically, the tumor-specific a and b TCR chains are expressed in lymphocytes that already bear an endogenous TCR on their cell surfaces. Gene-modified cells thus express at least two different TCRs that compete for binding to the CD3 complex, resulting in mutual TCR dilution and reduced avidity. Furthermore, since TCRs are heterodimers, the a and b chains of the endogenous TCR have the potential to mispair with the respective α and β chains of the transgenic TCR to produce a new hybrid TCR, with unpredictable and potentially harmful specificity. This represents a major concern in TCR transfer adoptive immunotherapy, both in autologous and allogeneic settings. To permanently eliminate the expression of the endogenous TCR and the risk of mispairing, our group recently developed a TCR gene editing approach. This technique is based on the transient transfer of zinc-finger nucleases (ZFN) to induce DNA double strand breaks in the constant regions of the endogenous TCR a and/or b chain genes, leading to permanent gene disruption. Upon lentiviral transfer of a tumor-specific TCR, such fully TCR-edited T cells express only the exogenous tumor-specific TCR transgenes at high levels (Provasi, Genovese et al., Nature Medicine 2012). While the complete editing procedure (both a and b TCR chains) currently requires multiple manipulation steps, ‘single TCR editing’, based on the ZFN-mediated knock-down of a single endogenous TCR chain (a or b) followed by the introduction of the tumor-specific TCR, enables the generation of redirected T cells devoid of their natural TCR repertoire during a single round of T cell activation, improving the feasibility of the clinical translation of this approach. This might be particularly useful to reduce the risk of GvHD after allogeneic hematopoietic stem cell transplantation. We exploited a HLA-A2 restricted TCR specific for NY-ESO-1, a cancer testis antigen expressed by solid tumors and hematological malignancies, to directly compare the safety and efficacy profile of unedited TCR transferred T cells (TR), single TCR edited (SE) lymphocytes and completely TCR edited (CE) T cells. We observed that gene editing does not detectably affect the phenotype, function or proliferative potential of engineered lymphocytes. Our protocols ensured the maintenance of the early differentiated memory phenotype, with enrichment in central memory and CD45RA+/CD62L+/CD95+ memory stem T (TSCM) cells. Upon lentiviral transfer of the NY-ESO-1-specific TCR, we observed significantly higher levels of the tumor-specific TCR expression, evaluated as NY-ESO-1 specific dextramer binding, in edited versus transferred T cells (relative fluorescence intensity to untransduced cells: CE: 37; SE: 31; TR: 19). Edited T cells were more efficient than unedited-TCR transferred T cells in killing NY-ESO-1-pulsed cell lines (half maximal effective peptide concentration in a 51Cr release assay: 310, 210, 186 nM for TR, SE and CE T cells respectively) and NY-ESO-1+ myeloma cell lines naturally processing the antigen. Importantly our SE and CE T cells displayed no activity against NY-ESO-1- targets. Importantly, in NSG mice, NY-ESO-1 redirected single edited and complete edited T cells completely eliminated an NY-ESO1+ HLA-A2+, WT1- myeloma cell line, that, on the contrary, expanded in bone marrow in the presence of WT1-redirected CE T cells. Our results demonstrate that the TCR single editing approach is effective in redirecting T cell specificity as evidenced by the potent anti-tumor effect observed while potentially eliminating the risk of GvHD associated with the infusion of donor-derived lymphocytes. Moreover, the relative speed and simplicity of the TCR single editing protocol should facilitate its clinical application to patients with hematological malignancies. Disclosures: Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

scholarly journals Probiotic supplementation reduces inflammatory profiles but does not prevent oral immune perturbations during SIV infection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rhianna Jones ◽  
Kyle Kroll ◽  
Courtney Broedlow ◽  
Luca Schifanella ◽  
Scott Smith ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Rhesus Macaques ◽  
Oral Microbiome ◽  
Probiotic Supplementation ◽  
Siv Infection ◽  
Anti Inflammatory

AbstractHIV/SIV infections lead to massive loss of mucosal CD4 + T cells and breakdown of the epithelial mucosa resulting in severe microbial dysbiosis and chronic immune activation that ultimately drive disease progression. Moreover, disruption of one of the most understudied mucosal environments, the oral cavity, during HIV-induced immunosuppression results in significant microbial and neoplastic co-morbidities and contributes to and predicts distal disease complications. In this study we evaluated the effects of oral probiotic supplementation (PBX), which can stimulate and augment inflammatory or anti-inflammatory pathways, on early SIV infection of rhesus macaques. Our study revealed that similar to the GI mucosae, oral CD4 + T cells were rapidly depleted, and as one of the first comprehensive analyses of the oral microflora in SIV infection, we also observed significant modulation among two genera, Porphyromonas and Actinobacillus, early after infection. Interestingly, although PBX therapy did not substantially protect against oral dysbiosis or ameliorate cell loss, it did somewhat dampen inflammation and T cell activation. Collectively, these data provide one of the most comprehensive evaluations of SIV-induced changes in oral microbiome and CD4 + T cell populations, and also suggest that oral PBX may have some anti-inflammatory properties in lentivirus infections.

scholarly journals Controlling T cells spreading, mechanics and activation by micropatterning

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anaïs Sadoun ◽  
Martine Biarnes-Pelicot ◽  
Laura Ghesquiere-Dierickx ◽  
Ambroise Wu ◽  
Olivier Théodoly ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Young Modulus ◽  
Careful Examination ◽  
Adhesion Receptor ◽  
Force Microscopy ◽  
Atomic Force ◽  
Micro Patterns

AbstractWe designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.

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