scholarly journals Deciphering Repertoire of B16 Melanoma Reactive TCRs by Immunization, In Vitro Restimulation and Sequencing of IFNγ-Secreting T Cells

2021 ◽  
Vol 22 (18) ◽  
pp. 9859
Author(s):  
Anna V. Izosimova ◽  
Diana V. Yuzhakova ◽  
Valeria D. Skatova ◽  
Lilia N. Volchkova ◽  
Elena V. Zagainova ◽  
...  
Keyword(s):  
Cell Receptor ◽  
B16 Melanoma ◽  
Adoptive Cell Transfer ◽  
Limiting Factors ◽  
Great Promise ◽  
T Helper ◽  
Cell Therapies ◽  
Optimized Protocol ◽  
Tumor Specificity

Recent advances in cancer immunotherapy have great promise for the treatment of solid tumors. One of the key limiting factors that hamper the decoding of physiological responses to these therapies is the inability to distinguish between specific and nonspecific responses. The identification of tumor-specific lymphocytes is also the most challenging step in cancer cell therapies such as adoptive cell transfer and T cell receptor (TCR) cloning. Here, we have elaborated a protocol for the identification of tumor-specific T lymphocytes and the deciphering of their repertoires. B16 melanoma engraftment following anti-PD1 checkpoint therapy provides better antitumor immunity compared to repetitive immunization with heat-shocked tumor cells. We have also revealed that the most error-prone part of dendritic cell (DC) generation, i.e., their maturation step, can be omitted if DCs are cultured at a sufficiently high density. Using this optimized protocol, we have achieved a robust IFNγ response to B16F0 antigens, but only within CD4+ T helper cells. A comparison of the repertoires of IFNγ-positive and -negative cells shows a prominent enrichment of certain clones with putative tumor specificity among the IFNγ+ fraction. In summary, our optimized protocol and the data provided here will aid in the acquisition of broad statistical data and the creation of a meaningful database of B16-specific TCRs.

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

scholarly journals Engineering Strategies to Enhance TCR-Based Adoptive T Cell Therapy

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1485 ◽  
Author(s):  
Jan A. Rath ◽  
Caroline Arber
Keyword(s):  
T Cell ◽  
Cell Receptor ◽  
Clinical Results ◽  
Adoptive T Cell Therapy ◽  
Great Promise ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Challenges And Opportunities ◽  
The Impact ◽  
Insight Into

T cell receptor (TCR)-based adoptive T cell therapies (ACT) hold great promise for the treatment of cancer, as TCRs can cover a broad range of target antigens. Here we summarize basic, translational and clinical results that provide insight into the challenges and opportunities of TCR-based ACT. We review the characteristics of target antigens and conventional αβ-TCRs, and provide a summary of published clinical trials with TCR-transgenic T cell therapies. We discuss how synthetic biology and innovative engineering strategies are poised to provide solutions for overcoming current limitations, that include functional avidity, MHC restriction, and most importantly, the tumor microenvironment. We also highlight the impact of precision genome editing on the next iteration of TCR-transgenic T cell therapies, and the discovery of novel immune engineering targets. We are convinced that some of these innovations will enable the field to move TCR gene therapy to the next level.

scholarly journals Pathogen boosted adoptive cell transfer immunotherapy to treat solid tumors

2017 ◽  
Vol 114 (4) ◽  
pp. 740-745 ◽  
Author(s):  
Gang Xin ◽  
David M. Schauder ◽  
Weiqing Jing ◽  
Aimin Jiang ◽  
Nikhil S. Joshi ◽  
...  
Keyword(s):  
T Cells ◽  
Solid Tumors ◽  
Cd8 T Cells ◽  
Cell Receptor ◽  
Genetically Engineered ◽  
Adoptive Cell Transfer ◽  
Bacterial Antigen ◽  
Cell Transfer ◽  
Primary Tumors

Because of insufficient migration and antitumor function of transferred T cells, especially inside the immunosuppressive tumor microenvironment (TME), the efficacy of adoptive cell transfer (ACT) is much curtailed in treating solid tumors. To overcome these challenges, we sought to reenergize ACT (ReACT) with a pathogen-based cancer vaccine. To bridge ACT with a pathogen, we genetically engineered tumor-specific CD8 T cells in vitro with a second T-cell receptor (TCR) that recognizes a bacterial antigen. We then transferred these dual-specific T cells in combination with intratumoral bacteria injection to treat solid tumors in mice. The dual-specific CD8 T cells expanded vigorously, migrated to tumor sites, and robustly eradicated primary tumors. The mice cured from ReACT also developed immunological memory against tumor rechallenge. Mechanistically, we have found that this combined approach reverts the immunosuppressive TME and recruits CD8 T cells with an increased number and killing ability to the tumors.

scholarly journals T Cell Receptor–Induced Calcineurin Activation Regulates T Helper Type 2 Cell Development by Modifying the Interleukin 4 Receptor Signaling Complex

2000 ◽  
Vol 191 (11) ◽  
pp. 1869-1880 ◽  
Author(s):  
Masakatsu Yamashita ◽  
Makoto Katsumata ◽  
Makio Iwashima ◽  
Motoko Kimura ◽  
Chiori Shimizu ◽  
...  
Keyword(s):  
Cell Receptor ◽  
Cell Development ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
T Helper ◽  
Signaling Complex ◽  
Th2 Cell ◽  
Interleukin 4 Receptor

The activation of downstream signaling pathways of both T cell receptor (TCR) and interleukin 4 receptor (IL-4R) is essential for T helper type 2 (Th2) cell development, which is central to understanding immune responses against helminthic parasites and in allergic and autoimmune diseases. However, little is known about how these two distinct signaling pathways cooperate with each other to induce Th2 cells. Here, we show that successful Th2 cell development depends on the effectiveness of TCR-induced activation of calcineurin. An inhibitor of calcineurin activation, FK506, inhibited the in vitro anti-TCR–induced Th2 cell generation in a dose-dependent manner. Furthermore, the development of Th2 cells was significantly impaired in naive T cells from dominant-negative calcineurin Aα transgenic mice, whereas that of Th1 cells was less affected. Efficient calcineurin activation in naive T cells upregulated Janus kinase (Jak)3 transcription and the amount of protein. The generation of Th2 cells induced in vitro by anti-TCR stimulation was inhibited significantly by the presence of Jak3 antisense oligonucleotides, suggesting that the Jak3 upregulation is an important event for the Th2 cell development. Interestingly, signal transducer and activator of transcription (STAT)5 became physically and functionally associated with the IL-4R in the anti-TCR–activated developing Th2 cells that received efficient calcineurin activation, and also in established cloned Th2 cells. In either cell population, the inhibition of STAT5 activation resulted in a diminished IL-4–induced proliferation. Moreover, our results suggest that IL-4–induced STAT5 activation is required for the expansion process of developing Th2 cells. Thus, Th2 cell development is controlled by TCR-mediated activation of the Ca2+/calcineurin pathway, at least in part, by modifying the functional structure of the IL-4R signaling complex.

scholarly journals T cell genetic background determines default T helper phenotype development in vitro.

1995 ◽  
Vol 181 (2) ◽  
pp. 713-721 ◽  
Author(s):  
C S Hsieh ◽  
S E Macatonia ◽  
A O'Garra ◽  
K M Murphy
Keyword(s):  
T Cells ◽  
T Cell ◽  
Genetic Background ◽  
Leishmania Major ◽  
Cell Receptor ◽  
T Helper ◽  
Alpha Beta ◽  
Development In Vitro

A host's ability to resist certain pathogens such as Leishmania major can depend upon the phenotype of T helper (Th) subset that develops. Different murine genetic backgrounds are known to significantly alter the direction of Th subset development, although the cellular basis of this influence is poorly understood. To examine the basis of this effect we used an in vitro alpha/beta-T cell receptor (TCR) transgenic system for analysis of Th phenotype development. To control for TCR usage, we derived the DO11.10 alpha/beta-TCR transgene in several genetic backgrounds. Our findings suggest that the effects of genetic background on Th phenotype development reside within the T cell, and not the antigen-presenting cell compartment. Transgenic T cells from both the B10.D2 and BALB/c backgrounds showed development toward either the Th1 or Th2 phenotype under the strong directing influence of interleukin (IL) 12 and IL4, respectively. However, when T cells were activated in vitro under neutral conditions in which exogenous cytokines were not added, B10.D2-derived T cells acquired a significantly stronger Th1 phenotype than T cells from the BALB/c background, correspondent with in vivo Th responses to Leishmania in these strains. Importantly, these cytokine differences resulted in distinct functional properties, because B10.D2- but not BALB/c-derived T cells could induce macrophage production of nitric oxide, an important antimicrobial factor. Thus, the genetically determined default Th phenotype development observed in vitro may correspond to in vivo Th subset responses for pathogens such as Leishmania which do not initiate strong Th phenotype-directing signals.

scholarly journals Immune deviation of 2C transgenic intraepithelial lymphocytes in antigen-bearing hosts.

1996 ◽  
Vol 184 (2) ◽  
pp. 493-503 ◽  
Author(s):  
S R Guehler ◽  
J A Bluestone ◽  
T A Barrett
Keyword(s):  
Cell Receptor ◽  
Intraepithelial Lymphocytes ◽  
Mrna Levels ◽  
T Helper ◽  
T Helper 1 ◽  
Ifn Gamma ◽  
Mrna Induction ◽  
Alpha Beta ◽  
Low Levels

The present study examined self-tolerance for T cell receptor (TCR) alpha beta intestinal intraepithelial lymphocytes (iIELs) using the 2C transgenic (Tg) mouse model specific for a peptide antigen (Ag) presented by the class I major histocompatibility complex H-2Ld. Although Tg+ T cells were largely deleted from the periphery of Ag+ mice, equivalent numbers of Tg iIELs were present in Ag+ compared to Ag- mice. Tg iIELs in Ag- mice contained CD8 alpha beta, CD8 alpha alpha, and CD4-CD8- subsets, whereas only CD8 alpha alpha and CD4-CD8- Tg iIEL subsets were detected in Ag+ mice. Analysis of surface markers revealed that Tg iIELs in Ag+ mice expressed decreased levels of Thy-1 and increased CD45R/B220 as compared to Ag- Tg iIELs. In response to activation with exogenous peptide or immobilized anti-TCR mAB, iIELs from Ag- mice proliferated at high levels and produced interleukin (IL)-2 and interferon (IFN)-gamma, while Tg+ iIELs from Ag+ mice proliferated at low levels and failed to produce detectable IL-2 or IFN-gamma. Activation of sorted iIEL subsets from Ag- mice revealed that CD8 alpha alpha and CD4-CD8- subsets produced low levels of IL-2 and IFN-gamma in response to activation with antigen-presenting cells and added peptide or immobilized anti-TCR mAb, while CD8 alpha beta + iIELs responded to endogenous levels of peptide. In response to APC and exogenous peptide, sorted iIEL subsets from Ag+ mice produced IL-2 and IFN-gamma, and proliferated at greatly reduced levels compared to corresponding subsets from Ag- mice. Analysis of cytokine mRNA levels revealed that activation in vitro induced IL-2 mRNA only in Ag-, but not Ag+ iIELs, whereas a high level of IL-4 mRNA induction was detected in Tg+ iIELs from Ag+ mice, and to a lesser degree, from Ag- mice. These data suggest that tolerance for Tg+ iIELs resulted in the deletion of CD8 alpha beta + subsets and the persistence of Tg+ iIEL subsets with decreased sensitivity to endogenous levels of self-peptide. A comparison of the cytokine profiles expressed by Tg+ iIEL subsets in Ag- and Ag+ mice suggested that tolerance induction had involved the functional deviation of cells from TC1 (T helper-1-like) to a less inflammatory TC2 (T helper-2-like) phenotype capable of mediating humoral immune responses in the mucosa.

scholarly journals 203 A membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence and efficacy of CD70-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A214-A214
Author(s):  
Jian Ding ◽  
Lindsay Webb ◽  
Troy Patterson ◽  
Michelle Fleury ◽  
Adam Zieba ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Hematological Malignancies ◽  
Cell Receptor ◽  
Target Cells ◽  
Cell Therapies ◽  
Anti Tumor Activity

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies. To realize the potential of T cell therapies in solid tumors, we have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes all TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. Previously, we have described the discovery and preclinical efficacy of fratricide-resistant TRuC-T cells targeting CD70, a tumor antigen overexpressed in various solid and hematological malignancies. As a strategy to enhance T cell effector function and persistence in the hostile tumor microenvironment, we engineered anti-CD70 TRuC-T cells to co-express a membrane-bound IL15Ra-IL15 fusion protein (IL-15fu). IL-15 is a common ? chain cytokine that promotes the differentiation, maintenance, and effector function of memory CD8+ T cell subsets and confers resistance to IL-2-mediated activation induced cell death (AICD).MethodsT cells were activated by CD3/CD28 stimulation and lentivirally transduced with a T2A-containing bicistronic vector encoding the anti-CD70 CD3?-TRuC and the IL-15fu proteins; the cells were further expanded for 9 days in media containing IL-7/IL-15. Surface co-expression of the TRuC and IL-15fu proteins and the T cell memory phenotype was assessed by flow cytometry. In vitro persistence was tested in a repeated stimulation assay in which T cells were challenged by addition of fresh CD70+ target cells every four days with longitudinal assessment of T-cell expansion, phenotype, cytokine production, and cytotoxicity. In vivo, the antitumor efficacy of the anti-CD70 TRuC/IL-15fu T cells was evaluated in MHC class I/II deficient NSG mice bearing human tumor xenografts.ResultsThe anti-CD70 TRuC and IL-15fu proteins showed high transduction efficiency and robust co-expression on the surface of T cells. The IL-15fu significantly increased the proportion of naïve cells within the TRuC-T cell product, most dramatically in the CD8+ subset. In vitro, TRuC-T cells bearing the IL-15fu showed greatly enhanced expansion and persistence upon repeated stimulation with CD70+ target cells. Moreover, the IL-15fu enhanced T-cell survival and persistence under unstimulated, cytokine-free conditions. In vivo, the anti-tumor activity of CD70-targeted TRuC-T cells was significantly improved by IL-15fu in multiple tumor models and was associated with enhanced intratumoral T-cell accumulation and a preferential expansion of CD8+ T cells.ConclusionsThe addition of the IL-15fu improved the phenotype, persistence, and anti-tumor activity of CD70-targeted TRuC-T cells, potentially increasing the likelihood of clinical benefit in patients with CD70 overexpressing solid and liquid cancers.Ethics ApprovalAll animal studies were conducted by TCR2 Therapeutics staff at the Charles River Laboratories CRADL facility under a protocol approved by the Charles River Laboratories Institutional Animal Care and Use Committee.

scholarly journals Programming tumor-reactive effector memory CD8+ T cells in vitro obviates the requirement for in vivo vaccination

Blood ◽  
2009 ◽  
Vol 114 (9) ◽  
pp. 1776-1783 ◽  
Author(s):  
Christopher A. Klebanoff ◽  
Zhiya Yu ◽  
Leroy N. Hwang ◽  
Douglas C. Palmer ◽  
Luca Gattinoni ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cell Receptor ◽  
Adoptive Cell Transfer ◽  
Effector Memory ◽  
Cell Transfer ◽  
Memory Cd8 T Cells ◽  
Ifn Γ

Abstract Naive and memory CD8+ T cells can undergo programmed activation and expansion in response to a short T-cell receptor stimulus, but the extent to which in vitro programming can qualitatively substitute for an in vivo antigen stimulation remains unknown. We show that self-/tumor-reactive effector memory CD8+ T cells (TEM) programmed in vitro either with peptide-pulsed antigen-presenting cells or plate-bound anti-CD3/anti-CD28 embark on a highly stereotyped response of in vivo clonal expansion and tumor destruction nearly identical to that of vaccine-stimulated TEM cells. This programmed response was associated with an interval of antigen-independent interferon-γ (IFN-γ) release that facilitated the dynamic expression of the major histocompatibility complex class I restriction element H-2Db on responding tumor cells, leading to recognition and subsequent tumor lysis. Delaying cell transfer for more than 24 hours after stimulation or infusion of cells deficient in IFN-γ entirely abrogated the benefit of the programmed response, whereas transfer of cells unable to respond to IFN-γ had no detriment to antitumor immunity. These findings extend the phenomenon of a programmable effector response to memory CD8+ T cells and have major implications for the design of current adoptive-cell transfer trials.

Type I Interferons promote maintenance and function of follicular T helper cells in vitro

2019 ◽  
Author(s):  
S Ehrlich ◽  
K Wild ◽  
M Smits ◽  
K Zoldan ◽  
M Hofmann ◽  
...  
Keyword(s):  
T Helper Cells ◽  
Type I Interferons ◽  
Type I ◽  
T Helper ◽  
Helper Cells ◽  
Follicular T Helper Cells ◽  
And Function

Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

2020 ◽  
Vol 16 ◽  
Author(s):  
Bruna O. S. Câmara ◽  
Bruno M. Bertassoli ◽  
Natália M. Ocarino ◽  
Rogéria Serakides
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture Medium ◽  
Adipogenic Differentiation ◽  
Medium Composition ◽  
Cell Therapies ◽  
Insulin Producing Cells ◽  
Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

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