Long-Term Maintenance In Vitro of Human T Cells by Repeated Exposure to the Same Stimulator Cells.

1975 ◽  
Vol 4 (5) ◽  
pp. 421-427 ◽  
Author(s):  
E. SVEDMYR
Keyword(s):  
T Cells ◽  
Repeated Exposure ◽  
Human T Cells ◽  
Term Maintenance

scholarly journals Rapid and Efficient Gene Editing for Direct Transplantation of Naive Murine Cas9+ T Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Snigdha Majumder ◽  
Isabelle Jugovic ◽  
Domenica Saul ◽  
Luisa Bell ◽  
Nadine Hundhausen ◽  
...  
Keyword(s):  
T Cells ◽  
Gene Editing ◽  
Metabolic Reprogramming ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Ribonucleoprotein Complexes ◽  
Human T Cells ◽  
Efficient Gene

Gene editing of primary T cells is a difficult task. However, it is important for research and especially for clinical T-cell transfers. CRISPR/Cas9 is the most powerful gene-editing technique. It has to be applied to cells by either retroviral transduction or electroporation of ribonucleoprotein complexes. Only the latter is possible with resting T cells. Here, we make use of Cas9 transgenic mice and demonstrate nucleofection of pre-stimulated and, importantly, of naive CD3+ T cells with guideRNA only. This proved to be rapid and efficient with no need of further selection. In the mixture of Cas9+CD3+ T cells, CD4+ and CD8+ conventional as well as regulatory T cells were targeted concurrently. IL-7 supported survival and naivety in vitro, but T cells were also transplantable immediately after nucleofection and elicited their function like unprocessed T cells. Accordingly, metabolic reprogramming reached normal levels within days. In a major mismatch model of GvHD, not only ablation of NFATc1 and/or NFATc2, but also of the NFAT-target gene IRF4 in naïve primary murine Cas9+CD3+ T cells by gRNA-only nucleofection ameliorated GvHD. However, pre-activated murine T cells could not achieve long-term protection from GvHD upon single NFATc1 or NFATc2 knockout. This emphasizes the necessity of gene-editing and transferring unstimulated human T cells during allogenic hematopoietic stem cell transplantation.

627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A663-A663
Author(s):  
Keegan Cooke ◽  
Juan Estrada ◽  
Jinghui Zhan ◽  
Jonathan Werner ◽  
Fei Lee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Mouse Models ◽  
T Cell Activation ◽  
Phase 1 ◽  
Phase 1 Study ◽  
Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

scholarly journals Long‐term in vitro expansion ensures increased yield of central memory T cells as perspective for manufacturing challenges

2021 ◽  
Author(s):  
Stefanie Herda ◽  
Andreas Heimann ◽  
Benedikt Obermayer ◽  
Elisa Ciraolo ◽  
Stefanie Althoff ◽  
...  
Keyword(s):  
T Cells ◽  
Memory T Cells ◽  
Central Memory ◽  
Central Memory T Cells ◽  
In Vitro Expansion

108 MCY-M11, a CAR-PBMC cell product transiently expressing a mesothelin targeted mRNA CAR, exhibits desirable functional and immune phenotype attributed to sustained antitumor immunity in vitro

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A120-A120
Author(s):  
Sashi Kasimsetty ◽  
Himavanth Gatla ◽  
Dhana Chinnasamy
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Antitumor Immunity ◽  
Memory T Cells ◽  
Cell Populations ◽  
Epitope Spreading ◽  
Antitumor Response ◽  
Cell Product

BackgroundMCY-M11, an anti-mesothelin CAR (Meso-CAR) mRNA transfected PBMC cell product manufactured through <1 day-process is under clinical evaluation for the treatment of advanced ovarian cancer and peritoneal mesothelioma. In this in-vitro study, we characterized the phenotypic and functional status of immune cell populations in MCY-M11 and their possible role in antitumor immunity.MethodsMCY-M11 cell product were generated using unmanipulated healthy donor PBMCs (n=5) by transfection of Meso-CAR mRNA using MaxCyte’s proprietary Flow Electroporation® system. Frozen MCY-M11 cell product was thawed and cultured for 18 hours, then co-cultured with hMSLNneg or hMSLNpos human mesothelioma cell line, MSTO-211H, or stimulated with anti-CD3/anti-CD28 antibodies in vitro for 8 days. Distinct cell populations in MCY-M11 were evaluated for kinetics and duration of CAR expression, differentiation, activation, exhaustion, and their ability to secrete various immunomodulatory molecules during in vitro stimulation. Antigen-specific proliferation and cytotoxicity of MCY-M11 against hMSLNpos tumor cells as well as their ability to mount long-term antitumor immunity through epitope spreading mechanisms were studied.ResultsIndividual cell populations in MCY-M11 exhibited a consistent but transient Meso-CAR expression persisting for about 7 days. Cell subsets in MCY-M11 acquired early signs of activation and differentiation within 18–24 hours post-culture, but only attained full activation and lineage-specific differentiation upon specific response to hMSLNpos tumor cells. hMSLN antigen experienced MCY-M11 retained significant fractions of Naïve and Central Memory T cells and increased percentage of Effector Memory T cells along with increased expression of CD62L, CD27, and chemokine receptors (CCR5, CCR7, and CXCR3). MCY-M11 exhibited strong antigen-specific cytotoxicity against hMSLNpos tumor cells with corresponding increase in activation and proliferation of CD4+ and CD8+ T cell subsets and displayed low or no acquisition of known exhaustion markers. NK cells also exhibited a functionally superior molecular signature exhibiting increased levels of NKG2D, NKp44, NKp46, FAS, and TRAIL. The Monocytes and B cells in MCY-M11 also acquired an activated, differentiated, and mature phenotype, expressing molecules required for antigen presentation (HLA-DR, HLA-ABC, and CD205) and T cell co-stimulation (CD80 and CD86) to mount a strong antitumor response. These phenotypic changes in cell subsets of MCY-M11 transpired with simultaneous secretion of potent immunostimulatory molecules and chemokines facilitating an extended antitumor response through epitope spreading.ConclusionsWe demonstrated that MCY-M11 is a unique cell product possessing a complete built-in immune cellular machinery with favorable phenotype and enhanced functions specialized in mediating an effective and long-term antitumor response.Trial RegistrationNCT03608618

scholarly journals CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection

2021 ◽  
Vol 22 (2) ◽  
pp. 912
Author(s):  
Nabila Seddiki ◽  
John Zaunders ◽  
Chan Phetsouphanh ◽  
Vedran Brezar ◽  
Yin Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Proportion ◽  
Long Term Memory ◽  
Ki 67 ◽  
Memory Cd4 T Cells ◽  
Hiv 1

HIV-1 infection rapidly leads to a loss of the proliferative response of memory CD4+ T lymphocytes, when cultured with recall antigens. We report here that CD73 expression defines a subset of resting memory CD4+ T cells in peripheral blood, which highly express the α-chain of the IL-7 receptor (CD127), but not CD38 or Ki-67, yet are highly proliferative in response to mitogen and recall antigens, and to IL-7, in vitro. These cells also preferentially express CCR5 and produce IL-2. We reasoned that CD73+ memory CD4+ T cells decrease very early in HIV-1 infection. Indeed, CD73+ memory CD4+ T cells comprised a median of 7.5% (interquartile range: 4.5–10.4%) of CD4+ T cells in peripheral blood from healthy adults, but were decreased in primary HIV-1 infection to a median of 3.7% (IQR: 2.6–6.4%; p = 0.002); and in chronic HIV-1 infection to 1.9% (IQR: 1.1–3%; p < 0.0001), and were not restored by antiretroviral therapy. Moreover, we found that a significant proportion of CD73+ memory CD4+ T cells were skewed to a gut-homing phenotype, expressing integrins α4 and β7, CXCR3, CCR6, CD161 and CD26. Accordingly, 20% of CD4+ T cells present in gut biopsies were CD73+. In HIV+ subjects, purified CD73+ resting memory CD4+ T cells in PBMC were infected with HIV-1 DNA, determined by real-time PCR, to the same level as for purified CD73-negative CD4+ T cells, both in untreated and treated subjects. Therefore, the proliferative CD73+ subset of memory CD4+ T cells is disproportionately reduced in HIV-1 infection, but, unexpectedly, their IL-7 dependent long-term resting phenotype suggests that residual infected cells in this subset may contribute significantly to the very long-lived HIV proviral DNA reservoir in treated subjects.

Growth of Human T-Cells Following in vitro Allograft Sensitization

1980 ◽  
pp. 269-273 ◽  
Author(s):  
J. L. Strausser ◽  
A. Rosenberg
Keyword(s):  
T Cells ◽  
Human T Cells

Keeping the beat. Focus on “Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro”

2012 ◽  
Vol 303 (12) ◽  
pp. C1218-C1219 ◽  
Author(s):  
John P. Konhilas ◽  
Samantha M. Behunin ◽  
Ronald M. Lynch
Keyword(s):  
Primary Culture ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Neonatal Rat Cardiomyocytes ◽  
Term Maintenance

scholarly journals Pharmacologic marrow purging in murine T cell leukemia

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1656-1661 ◽  
Author(s):  
EA Copelan ◽  
SC Johnson ◽  
MR Grever ◽  
JF Sheridan ◽  
PJ Tutschka
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
In Vitro Assays ◽  
Cell Leukemia ◽  
Term Survival ◽  
Adenosine Deaminase Activity ◽  
Marrow Purging ◽  
Malignant T Cells

Abstract Deoxycoformycin in combination with deoxyadenosine was used to purge 6C3HED malignant T cells from murine marrow in vitro. Adenosine deaminase activity of 6C3HED cells was ablated by incubation with 10(- 6) mol/L deoxycoformycin (dCF). During a 12-hour incubation with 10(-6) mol/L dCF and 10(-4) mol/L deoxyadenosine, tumor cells sequentially accumulated dATP, became depleted of NAD followed by ATP, then died. More than 5 logs of 6C3HED cells were killed as measured by survival of mice injected with treated tumor cells. Identical incubation of 5 x 10(6) marrow cells did not interfere with rescue of syngeneic lethally irradiated mice. Long-term survival was demonstrated in 12 of 14 mice that received marrow that had been contaminated with 5% 6C3HED cells, incubated with deoxycoformycin and deoxyadenosine, then used to rescue lethally irradiated mice. This murine model provides information not available from in vitro assays and may be useful in the development of strategies to purge malignant T cells from marrow.

scholarly journals Melatonin Synergizes With Mesenchymal Stromal Cells Attenuates Chronic Allograft Vasculopathy

2021 ◽  
Vol 12 ◽  
Author(s):  
Ya-fei Qin ◽  
De-jun Kong ◽  
Hong Qin ◽  
Yang-lin Zhu ◽  
Guang-ming Li ◽  
...  
Keyword(s):  
T Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Th17 Cells ◽  
Memory T Cells ◽  
Transplant Recipients ◽  
Pathological Changes ◽  
Allograft Vasculopathy

BackgroundChronic rejection characterized by chronic allograft vasculopathy (CAV) remains a major obstacle to long-term graft survival. Due to multiple complicated mechanisms involved, a novel therapy for CAV remains exploration. Although mesenchymal stromal cells (MSCs) have been ubiquitously applied to various refractory immune-related diseases, rare research makes a thorough inquiry in CAV. Meanwhile, melatonin (MT), a wide spectrum of immunomodulator, plays a non-negligible role in transplantation immunity. Here, we have investigated the synergistic effects of MT in combination with MSCs in attenuation of CAV.MethodsC57BL/6 (B6) mouse recipients receiving BALB/c mouse donor aorta transplantation have been treated with MT and/or adipose-derived MSCs. Graft pathological changes, intragraft immunocyte infiltration, splenic immune cell populations, circulating donor-specific antibodies levels, cytokine profiles were detected on post-operative day 40. The proliferation capacity of CD4+ and CD8+ T cells, populations of Th1, Th17, and Tregs were also assessed in vitro.ResultsGrafts in untreated recipients developed a typical pathological feature of CAV characterized by intimal thickening 40 days after transplantation. Compared to untreated and monotherapy groups, MT in combination with MSCs effectively ameliorated pathological changes of aorta grafts indicated by markedly decreased levels of intimal hyperplasia and the infiltration of CD4+ cells, CD8+ cells, and macrophages, but elevated infiltration of Foxp3+ cells. MT either alone or in combination with MSCs effectively inhibited the proliferation of T cells, decreased populations of Th1 and Th17 cells, but increased the proportion of Tregs in vitro. MT synergized with MSCs displayed much fewer splenic populations of CD4+ and CD8+ T cells, Th1 cells, Th17 cells, CD4+ central memory T cells (Tcm), as well as effector memory T cells (Tem) in aorta transplant recipients. In addition, the percentage of splenic Tregs was substantially increased in the combination therapy group. Furthermore, MT combined with MSCs markedly reduced serum levels of circulating allospecific IgG and IgM, as well as decreased the levels of pro-inflammatory IFN-γ, TNF-α, IL-1β, IL-6, IL-17A, and MCP-1, but increased the level of IL-10 in the recipients.ConclusionsThese data suggest that MT has synergy with MSCs to markedly attenuate CAV and provide a novel therapeutic strategy to improve the long-term allograft acceptance in transplant recipients.

scholarly journals In vitro response of human T cells to Pseudomonas aeruginosa.

1983 ◽  
Vol 40 (2) ◽  
pp. 670-674 ◽  
Author(s):  
J M Porwoll ◽  
H M Gebel ◽  
G E Rodey ◽  
R B Markham
Keyword(s):  
Pseudomonas Aeruginosa ◽  
T Cells ◽  
Human T Cells ◽  
In Vitro Response
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