scholarly journals G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered TCR-T cells against hepatocellular carcinoma

2021 ◽  
Author(s):  
Maxine Lam ◽  
Jose Reales-Calderon ◽  
Jin Rong Ow ◽  
Joey AW ◽  
Damien TAN ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
T Cell Activation ◽  
Insertional Mutagenesis ◽  
Cell Activation ◽  
Ex Vivo ◽  
T Cell Therapy ◽  
Engineered T Cells

Abstract Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we found that short-term inhibition of G9a/GLP increased T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increased granzyme expression without terminal T cell differentiation or exhaustion and resulted in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

scholarly journals Optimizing Manufacturing Protocols of Chimeric Antigen Receptor T Cells for Improved Anticancer Immunotherapy

2019 ◽  
Vol 20 (24) ◽  
pp. 6223 ◽  
Author(s):  
Sophia Stock ◽  
Michael Schmitt ◽  
Leopold Sellner
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Chimeric Antigen Receptor ◽  
Cell Activation ◽  
Ex Vivo ◽  
Antigen Receptor ◽  
Tumor Control ◽  
Therapeutic Success ◽  
T Cell Therapy

Chimeric antigen receptor (CAR) T cell therapy can achieve outstanding response rates in heavily pretreated patients with hematological malignancies. However, relapses occur and they limit the efficacy of this promising treatment approach. The cellular composition and immunophenotype of the administered CART cells play a crucial role for therapeutic success. Less differentiated CART cells are associated with improved expansion, long-term in vivo persistence, and prolonged anti-tumor control. Furthermore, the ratio between CD4+ and CD8+ T cells has an effect on the anti-tumor activity of CART cells. The composition of the final cell product is not only influenced by the CART cell construct, but also by the culturing conditions during ex vivo T cell expansion. This includes different T cell activation strategies, cytokine supplementation, and specific pathway inhibition for the differentiation blockade. The optimal production process is not yet defined. In this review, we will discuss the use of different CART cell production strategies and the molecular background for the generation of improved CART cells in detail.

A Randomized Phase II Trial of Idiotype Vaccination and Adoptive Autologous T-Cell Transfer in Multiple Myeloma patients

Blood ◽  
2021 ◽  
Author(s):  
Muzaffar H Qazilbash ◽  
Neeraj Y Saini ◽  
Cha Soung-chul ◽  
Zhe Wang ◽  
Edward Stadtmauer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Phase Ii ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Phase Ii Trial ◽  
Ex Vivo ◽  
Vaccine Strategy ◽  
Randomized Phase Ii

We hypothesized that combining adoptively transferred autologous T cells with a cancer vaccine strategy would enhance therapeutic efficacy by adding anti-myeloma idiotype-keyhole limpet hemocyanin (Id-KLH) vaccine to vaccine-specific co-stimulated T cells. In this randomized, phase II trial, eligible patients received either the control (KLH only) or Id-KLH vaccine, an auto-transplant, vaccine-specific co-stimulated T-cells expanded ex-vivo, and two booster doses of the assigned vaccine. In 36 patients (20 in KLH, 16 in Id-KLH) enrolled, no dose-limiting toxicity was seen in either arm. At last evaluation, 6 (30%) and 8 (50%) had achieved complete remission in KLH-only and Id-KLH, respectively (p=0.22) and no difference in 3-year progression-free survival was observed (59% and 56%, respectively; p=0.32). In a 594 Nanostring nCounter gene panel analyzed for immune reconstitution (IR), compared with KLH-only patients, there was a greater change in IR genes in T-cells in Id-KLH patients relative to baseline. Specifically, upregulation of genes associated with activation, induction of effector function, and generation of memory CD8+ T cells after Id-KLH, but not after KLH control vaccination, was observed. Similarly, responding patients across both arms were associated with upregulation of genes associated with T-cell activation. At baseline, all patients had greater expression of CD8+ T-cell exhaustion markers. These changes were associated with functional Id-specific immune responses in a subset of Id-KLH patients analyzed. In conclusion, in this combination immunotherapy approach, we observed a significantly more robust IR in CD4+ and CD8+ T cells in the Id-KLH arm, supporting further investigation of vaccine and adoptive immunotherapy strategies.

scholarly journals Inconsistent Reversal of HIV-1 Latency Ex Vivo by Antigens of HIV-1, CMV, and Other Infectious Agents

2020 ◽  
Author(s):  
Thomas Vollbrecht ◽  
Aaron O. Angerstein ◽  
Bryson Menke ◽  
Nikesh M. Kumar ◽  
Michelli Faria Oliveira ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Latent Reservoir ◽  
Antigen Specificity ◽  
Pcr Assay ◽  
Hiv 1

Abstract BackgroundA reservoir of replication-competent but latent virus is the main obstacle to a cure for HIV-infection. Much of this reservoir resides in memory CD4 T cells. We hypothesized that these cells can be reactivated with antigens from HIV and other common pathogens to reverse latency. ResultsWe obtained mononuclear cells from the peripheral blood of antiretroviral-treated patients with suppressed viremia. We tested pools of peptides and proteins derived from HIV and from other pathogens including CMV for their ability to reverse latency ex vivo by activation of memory responses. We assessed activation of the CD4 T cells by measuring the up-regulation of cell-surface CD69. We assessed HIV-expression using two assays: a real-time PCR assay for virion-associated viral RNA and a droplet digital PCR assay for cell-associated, multiply spliced viral mRNA. Reversal of latency occurred in a minority of cells from some participants, but no single antigen induced HIV-expression ex vivo consistently. When reversal of latency was induced by a specific peptide pool or protein, the extent was proportionally greater than that of T cell activation. ConclusionsIn this group of patients in whom antiretroviral therapy was started during chronic infection, the latent reservoir does not appear to consistently reside in CD4 T cells of a predominant antigen-specificity. Peptide-antigens reversed HIV-latency ex vivo with modest and variable activity. When latency was reversed by specific peptides or proteins, it was proportionally greater than the extent of T cell activation, suggesting partial enrichment of the latent reservoir in cells of specific antigen-reactivity.

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

A class of costimulatory CD28-bispecific antibodies that enhance the antitumor activity of CD3-bispecific antibodies

2020 ◽  
Vol 12 (525) ◽  
pp. eaaw7888 ◽  
Author(s):  
Dimitris Skokos ◽  
Janelle C. Waite ◽  
Lauric Haber ◽  
Alison Crawford ◽  
Aynur Hermann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Specific Antigen ◽  
The Other ◽  
Antitumor Efficacy ◽  
Bispecific Antibodies

T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide–major histocompatibility complexes (“signal 1”); activation is enhanced by engagement of a second “costimulatory” receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell (“signal 2”). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.

scholarly journals HIV-specific CD8+ T-cells in tonsils express exhaustive TRM-like signatures

2021 ◽  
Author(s):  
Rabiah Fardoos ◽  
Sarah K. Nyquist ◽  
Osaretin E. Asowata ◽  
Samuel W. Kazer ◽  
Alveera Singh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Cytolytic Activity ◽  
Lymphoid Tissues ◽  
People Living With Hiv ◽  
Natural Immunity

Lymphoid tissues are an important HIV reservoir site that persists in the face of antiretroviral therapy and natural immunity. Targeting these reservoirs by harnessing the antiviral activity of local tissue resident memory (TRM) CD8+ T-cells is of great interest, but limited data exist on TRMs within lymph nodes of people living with HIV (PLWH). Here, we studied tonsil CD8+ T-cells obtained from PLWH and uninfected controls from South Africa. We show that these cells are preferentially located outside the germinal centers (GCs), the main reservoir site for HIV, and display a low cytolytic and transcriptionally TRM-like profile that is distinct from blood. In PLWH, CD8+ TRM-like cells are highly expanded and adopt a more cytolytic, activated and exhausted phenotype characterized by increased expression of CD69, PD-1 and perforin, but reduced CD127. This phenotype was enhanced in HIV-specific CD8+ T-cells from tonsils compared to matched blood. Single-cell profiling of these cells revealed a clear transcriptional signature of T-cell activation, clonal expansion and exhaustion ex-vivo. In contrast, this signature was absent from HIV-specific CD8+ T-cells in tonsils isolated from a natural HIV controller, who expressed lower levels of cell surface PD-1 and CXCR5, and reduced transcriptional evidence of T-cell activation, exhaustion and cytolytic activity. Thus, we show that HIV-specific TRM-like CD8+ T-cells in tonsils from non-HIV controllers are enriched for activation and exhaustion profiles compared to those in blood, suggesting that lymphoid HIV specific CD8+ TRM cells are potentially ideal candidates for immunotherapy to modulate their ability to targeting the HIV reservoirs.

scholarly journals Early T Cell Activation Correlates with Expression of Apoptosis Markers in Patients with End-Stage Renal Disease

2002 ◽  
Vol 13 (1) ◽  
pp. 204-212
Author(s):  
Pascal Meier ◽  
Eric Dayer ◽  
Edouard Blanc ◽  
Jean-Pierre Wauters
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dna Fragmentation ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Annexin V ◽  
Culture Conditions ◽  
T Cell Apoptosis ◽  
End Stage

ABSTRACT. End-stage renal failure (ESRF) and chronic hemodialysis (HD) induce a state of immunodeficiency that involves T cell-mediated responses. A decreased T cell number combined with a reduced T cell lifespan and an increased T cell activation might play a role in the immune impairment associated with ESRF and chronic HD. Increased T cell activation associated with immunodeficiency suggests that activated T cells may be driven to apoptosis. To test this hypothesis, CD3+ T cell activation (CD69) and apoptosis (annexin V, CD95 (Fas), and DNA fragmentation) were analyzed in a case control study after blood draw sampling (ex vivo), in culture conditions, and after phytohemagglutinin or anti-CD3 stimulation. Ex vivo evaluation of T cells showed an increased number of activated CD69+ T cells in chronic HD patients (142 ± 5 cells/mm3) compared with patients with ESRF (115 ± 2 cells/mm3, P = 0.04) and controls (74 ± 2 cells/mm3, P = 0.0006). These data were confirmed in culture conditions and after stimulation. Similarly, annexin V and CD95 (Fas)-positive T cells were more numerous in both patient groups than in controls, irrespective of the experimental conditions (P ≤ 0.005 for both markers), and their percentage was always significantly higher in chronic HD patients than in patients with ESRF. The amount of DNA fragmentation was also significantly higher in the cultured resting T cells of chronic HD patients (37 ± 3%) than in those of patients with ESRF (25 ± 3%) and controls (20 ± 2%) (P = 0.01). Percentage of cultured resting T cells expressing both CD69 and annexin V markers was higher in chronic HD patients (17 ± 4%) than in patients with ESRF (10 ± 4%) and controls (6 ± 2%), (P = 0.005). After stimulation (phytohemagglutinin or anti-CD3), CD69+ T cell apoptosis increased by 2.4-fold in chronic HD patients compared with 1.8-fold in patients with ESRF and only 1.2-fold in controls (P = 0.001). T cells from chronic HD patients and patients with ESRF thus showed an aberrant state of early activation that contrasted with an increased proportion of annexin V and CD95 (Fas)-positive T cells engaged in apoptosis, as confirmed by DNA fragmentation. Increased susceptibility to early activated T cell apoptosis is not only associated with uremia, but is also enhanced by HD procedure. This may account for the T lymphopenia, progressive immunodeficiency, and increased infection risk seen in these patients.

scholarly journals Computational and experimental optimization of T cell activation

2019 ◽  
Author(s):  
Bulent Arman Aksoy ◽  
Eric Czech ◽  
Chrystal Paulos ◽  
Jeff Hammerbacher
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Experimental Investigations ◽  
Short Term ◽  
Cell Ratio ◽  
Culture Density

AbstractBead-based activation is widely-used for ex vivo expansion of T cells for either research or clinical purposes. Despite its wide use, culture conditions that can potentially affect the efficiency of bead-based T cell activation has not been extensively documented. With the help of computationally-driven experimental investigations of basic culturing factors, we found that culture density, bead-to-cell ratio, and debeading time can have a major impact on the efficiency of bead-based T cell activation for short-term cultures. Furthermore, discrepancies across expected and observed activation efficiencies helped discover interesting artifacts of bead-based T cell activation.Human primary T cells were imaged together with activation beads at 20X magnification after three hours of culturing at varying confluencies and bead-to-cell ratios.

scholarly journals Blinatumomab-Induced T Cell Activation at Single Cell Transcriptome Resolution

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3886-3886
Author(s):  
Hong Yin ◽  
Yi Huo ◽  
Zhen Sheng ◽  
Chi-Ming Li ◽  
Daniel C Ellwanger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Target Cells ◽  
Ex Vivo Model ◽  
Naive T Cells ◽  
Naïve T Cells

Introduction Blinatumomab, a bi-specific T cell engager (BiTE®) combining the VH and VL domains of two antibodies against human CD19 and CD3, has been approved by U.S. Food and Drug Administration (FDA) for the treatment of patients with relapsed or refractory B precursor ALL (r/r B-ALL) for its clinical benefit demonstrated in different clinical trials. Clinical trials have also shown that there are still patients refractory to blinatumomab. It is thus important to understand the resistance mechanisms. Blinatumomab connects patients' autologous T cells and target cells to form immunologic synapse which potently triggers the activation signaling cascades in T cells and guides T cells to recognize and induce perforin/granzyme-mediated lysis of CD19+ B-ALL cells. Previous studies showed blinatumomab-mediated cytotoxicity involves different T cell subpopulations. But response of each T cell subpopulation to blinatumomab treatment remained largely unknown. Methods and Results In this study, we used 10X Genomics based single cell RNA sequencing to analyze the transcriptome of single T cells before and after blinatumomab treatment. First, ex vivo blinatumomab cytotoxicity model was established, in which healthy PBMCs were used as effector cells and cocultured with target cells (RS4;11 cells or SUP-B15 cells) at an effector-to-target cell ratio of 10:1 with 0.1 ng/mL blinatumomab. Then, PBMCs and BMMCs from 2 B-ALL patients were cultured with 10 ng/mL blinatumomab. Cells from both ex vivo model and patient samples were sequenced using 10X Genomics platform. In total, transcriptome of 17920 single T cells from the ex vivo model and 2271 single T cells from patient sample were analyzed. Based on T cell trajectory analysis, we identified four distinct populations of blinatumomab-activated T cells, which were derived from CD8+ effector memory T (TEM) cells, CD4+ central memory (TCM) cells, naïve T cells and Tregs, respectively. The differentially expressed genes in activated clusters were analyzed to reflect T cell activation mechanisms. The result showed blinatumomab induced the upregulation of aerobic glycolysis pathway (PKM, PGAM1, ENO1, GAPDH and LDHA), cytoskeleton dynamics pathway (ACTD1, ACTB, NME1 and TUBA1B), IFN-responsive pathway (GBP1, PSME2, WARS, CXCL10 and STAT1), and the upregulation of well-known immune-related genes (TNFRSF4, TNFRSF18, LAG3, CD69, IL2RA, MIR155HG, BATF, SH2D2A, LTA, NFKBIA and NDFIP2). We found blinatumomab-activated CD8+ TEM cells showed stronger cytotoxic capability than other activated populations with specific production of cytotoxic factors (PRF1, IFNG and FASLG) and cytokines (CCL2, CCL3, CCL3L1, CCL4, CCL4L2, CCL8, XCL1, XCL2, TNFSF9 and TNFSF14). Last, differential gene expression analysis revealed that co-stimulatory (TNFRSF4,TNFRSF9 and TNFRSF18) and co-inhibitory receptors (LAG3 and TIGIT) were similarly up-regulated in clusters activated from memory and naïve T cells, indicating ligand dependent T cell functional outcomes induced by blinatumomab. Conclusion In summary, we used single cell sequencing to map the blinatumomab-mediated T cell activation state transition and reveal the molecular changes in different T cell subpopulations. Memory T cells, naïve T cells and Tregs were identified functional populations after blinatumomab treatment. CD8+ TEM accounted for the majority of blinatumomab-induced cytotoxicity. Furthermore, T cell co-regulatory receptors were identified as potential targets accountable for blinatumomab sensitivity or resistance mechanisms. The study demonstrated that the collected cellular transcriptional profiles can serve as resource to explore novel strategies to enhance the efficacy of blinatumomab. Disclosures Yin: Amgen: Employment. Huo:Amgen: Employment. Sheng:Amgen: Employment. Li:Amgen: Employment. Ellwanger:Amgen: Employment. Lu:Amgen: Employment. Homann:Amgen: Employment. Wang:Amgen: Employment. Ren:Ruijin hospital: Employment.

scholarly journals The S enantiomer of 2-hydroxyglutarate increases central memory CD8 populations and improves CAR-T therapy outcome

2020 ◽  
Vol 4 (18) ◽  
pp. 4483-4493
Author(s):  
Iosifina P. Foskolou ◽  
Laura Barbieri ◽  
Aude Vernet ◽  
David Bargiela ◽  
Pedro P. Cunha ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antitumor Activity ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Phase 1 ◽  
Therapeutic Success ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T

Abstract Cancer immunotherapy is advancing rapidly and gene-modified T cells expressing chimeric antigen receptors (CARs) show particular promise. A challenge of CAR-T cell therapy is that the ex vivo–generated CAR-T cells become exhausted during expansion in culture, and do not persist when transferred back to patients. It has become clear that naive and memory CD8 T cells perform better than the total CD8 T-cell populations in CAR-T immunotherapy because of better expansion, antitumor activity, and persistence, which are necessary features for therapeutic success and prevention of disease relapse. However, memory CAR-T cells are rarely used in the clinic due to generation challenges. We previously reported that mouse CD8 T cells cultured with the S enantiomer of the immunometabolite 2-hydroxyglutarate (S-2HG) exhibit enhanced antitumor activity. Here, we show that clinical-grade human donor CAR-T cells can be generated from naive precursors after culture with S-2HG. S-2HG–treated CAR-T cells establish long-term memory cells in vivo and show superior antitumor responses when compared with CAR-T cells generated with standard clinical protocols. This study provides the basis for a phase 1 clinical trial evaluating the activity of S-2HG–treated CD19-CAR-T cells in patients with B-cell malignancies.

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