T-cells with a single tumor antigen-specific T-cell receptor can be generated in vitro from clinically relevant stem cell sources

Intracellular signaling from the T cell receptor (TCR)zeta/CD3 complex is likely to be mediated by associated protein tyrosine kinases such as p59fyn(T), ZAP-70, and the CD4:p56lck and CD8:p56lck coreceptors. The nature of the signaling cascade initiated by these kinases, their specificities, and downstream targets remain to be elucidated. The TCR-zeta/CD3:p59fyn(T) complex has previously been noted to coprecipitate a 120/130-kD doublet (p120/130). This intracellular protein of unknown identity associates directly with p59fyn(T) within the receptor complex. In this study, we have shown that this interaction with p120/130 is specifically mediated by the SH2 domain (not the fyn-SH3 domain) of p59fyn(T). Further, based on the results of in vitro kinase assays, p120/130 appears to be preferentially associated with p59fyn(T) in T cells, and not with p56lck. Antibody reprecipitation studies identified p120/130 as a previously described 130-kD substrate of pp60v-src whose function and structure is unknown. TCR-zeta/CD3 induced activation of T cells augmented the tyrosine phosphorylation of p120/130 in vivo as detected by antibody and GST:fyn-SH2 fusion proteins. p120/130 represents the first identified p59fyn(T):SH2 binding substrate in T cells, and as such is likely to play a key role in the early events of T cell activation.

Download Full-text

CD4+ T Cells from Glutamic Acid Decarboxylase (GAD)65-specific T Cell Receptor Transgenic Mice Are Not Diabetogenic and Can Delay Diabetes Transfer

Journal of Experimental Medicine ◽

10.1084/jem.20011845 ◽

2002 ◽

Vol 196 (4) ◽

pp. 481-492 ◽

Cited By ~ 62

Author(s):

Kristin V. Tarbell ◽

Mark Lee ◽

Erik Ranheim ◽

Cheng Chi Chao ◽

Maija Sanna ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Transgenic Mice ◽

T Cell Receptor ◽

Glutamic Acid Decarboxylase ◽

Cd4 T Cells ◽

Cell Receptor ◽

Acid Decarboxylase ◽

Gad 65

Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286–300 (p286) of GAD65. These mice have GAD65-specific CD4+ T cells, as shown by staining with an I-Ag7(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, and IL-10 when stimulated in vitro with GAD65 peptide 286–300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4+ T cells, or p286-tetramer+CD4+ Tcells, from GAD65 286–300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286–300-specific T cells have disease protective capacity and are not pathogenic.

Download Full-text

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.173 ◽

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.

Download Full-text

BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to In Vitro Differentiation into Functional Foxp3+ T Regulatory Cells

PLoS ONE ◽

10.1371/journal.pone.0112242 ◽

2014 ◽

Vol 9 (11) ◽

pp. e112242 ◽

Cited By ~ 2

Author(s):

Ghanashyam Sarikonda ◽

Georgia Fousteri ◽

Sowbarnika Sachithanantham ◽

Jacqueline F. Miller ◽

Amy Dave ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd4 T Cells ◽

T Regulatory Cells ◽

Cell Receptor ◽

Regulatory Cells ◽

In Vitro Differentiation

Download Full-text

Cytomegalovirus-Specific CD8+ T-Cells With Different T-Cell Receptor Affinities Segregate T-Cell Phenotypes and Correlate With Chronic Graft-Versus-Host Disease in Patients Post-Hematopoietic Stem Cell Transplantation

Frontiers in Immunology ◽

10.3389/fimmu.2018.00760 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 4

Author(s):

Thomas Poiret ◽

Rebecca Axelsson-Robertson ◽

Mats Remberger ◽

Xiao-Hua Luo ◽

Martin Rao ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Cell Receptor ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

T Cell Phenotypes ◽

Cell Phenotypes

Download Full-text

T-cell receptor-α repertoire of CD8+ T cells following allogeneic stem cell transplantation using next-generation sequencing

Haematologica ◽

10.3324/haematol.2018.199802 ◽

2018 ◽

Vol 104 (3) ◽

pp. 622-631 ◽

Cited By ~ 9

Author(s):

Cornelia S. Link-Rachner ◽

Anne Eugster ◽

Elke Rücker-Braun ◽

Falk Heidenreich ◽

Uta Oelschlägel ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Next Generation Sequencing ◽

T Cell ◽

Stem Cell Transplantation ◽

T Cell Receptor ◽

Cd8 T Cells ◽

Allogeneic Stem Cell Transplantation ◽

Cell Receptor ◽

Generation Sequencing

Download Full-text

Prediction of T-cell reconstitution by assessment of T-cell receptor excision circle before allogeneic hematopoietic stem cell transplantation in pediatric patients

Blood ◽

10.1182/blood-2004-04-1405 ◽

2005 ◽

Vol 105 (2) ◽

pp. 886-893 ◽

Cited By ~ 52

Author(s):

Xiaohua Chen ◽

Raymond Barfield ◽

Ely Benaim ◽

Wing Leung ◽

James Knowles ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

T Cell Receptor ◽

Cell Receptor ◽

Hematopoietic Stem ◽

Thymic Function ◽

T Cell Reconstitution

Abstract The extent and rapidity with which T cells are regenerated from graft-derived precursor cells directly influences the incidence of infection and the T-cell–based graft-versus-tumor effect. Measurement of T-cell receptor excision circles (TRECs) in peripheral blood is a means of quantifying recent thymic T-cell production and has been used after transplantation in many studies to estimate thymus-dependent T-cell reconstitution. We hypothesized that the quality of thymic function before transplantation affects thymus-dependent T-cell reconstitution after transplantation. We used real-time polymerase chain reaction (PCR) to quantify signal-joint TRECs (sjTRECs) before and after transplantation. T-cell reconstitution was evaluated by T-cell receptor β (TCRβ) CDR3 size spectratyping. We tested 77 healthy sibling donors and 244 samples from 26 pediatric recipients of allogeneic hematopoietic stem cell transplantation (AHSCT). Blood from the healthy donors contained 1200 to 155 000 sjTREC copies/mL blood. Patients who had greater than 1200 copies/mL blood before transplantation showed early recovery of sjTREC numbers and TCRβ repertoire diversity. In contrast, patients who had fewer than 1200 copies/mL blood before transplantation demonstrated significantly slower restoration of thymus-dependent T cells. We conclude that the rate of reconstitution of thymus-dependent T cells is dependent on the competence of thymic function in the recipients before transplantation. Therefore, pretransplantation measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution after transplantation.

Download Full-text

Abstract A043: Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro

10.1158/2326-6074.cricimteatiaacr18-a043 ◽

2019 ◽

Author(s):

Dana Stenger ◽

Tanja Stief ◽

Theresa Käuferle ◽

Semjon Manuel Willier ◽

Felicitas Rataj ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Car T Cells ◽

Car T

Download Full-text

A Novel Approach for the Treatment of T Cell Malignancies: Targeting T Cell Receptor Vβ Families

Vaccines ◽

10.3390/vaccines8040631 ◽

2020 ◽

Vol 8 (4) ◽

pp. 631

Author(s):

Jie Wang ◽

Katarzyna Urbanska ◽

Prannda Sharma ◽

Reza Nejati ◽

Lauren Shaw ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

T Cell Lymphomas ◽

Novel Approach ◽

T Cell Malignancies

Peripheral T cell lymphomas (PTCLs) are generally chemotherapy resistant and have a poor prognosis. The lack of targeted immunotherapeutic approaches for T cell malignancies results in part from potential risks associated with targeting broadly expressed T cell markers, namely T cell depletion and clinically significant immune compromise. The knowledge that the T cell receptor (TCR) β chain in human α/β TCRs are grouped into Vβ families that can each be targeted by a monoclonal antibody can therefore be exploited for therapeutic purposes. Here, we develop a flexible approach for targeting TCR Vβ families by engineering T cells to express a chimeric CD64 protein that acts as a high affinity immune receptor (IR). We found that CD64 IR-modified T cells can be redirected with precision to T cell targets expressing selected Vβ families by combining CD64 IR-modified T cells with a monoclonal antibody directed toward a specific TCR Vβ family in vitro and in vivo. These findings provide proof of concept that TCR Vβ-family-specific T cell lysis can be achieved using this novel combination cell–antibody platform and illuminates a path toward high precision targeting of T cell malignancies without substantial immune compromise.

Download Full-text

Evidence for in vivo clonal proliferation of unique population of blood CD4-/CD8- T cells bearing T-cell receptor alpha and beta chains in two normal men

Blood ◽

10.1182/blood.v79.11.2965.2965 ◽

1992 ◽

Vol 79 (11) ◽

pp. 2965-2972 ◽

Cited By ~ 20

Author(s):

Y Kusunoki ◽

Y Hirai ◽

S Kyoizumi ◽

M Akiyama

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Nk Cell ◽

Cell Receptor ◽

Cell Markers ◽

Clonal Proliferation ◽

Alpha Beta

Abstract Rare T lymphocytes bearing CD3 surface antigen and T-cell receptor (TCR) alpha and beta chains, but lacking both CD4 and CD8 antigens, viz, TCR alpha beta+CD4–8- cells, appear at a frequency of 0.1% to 2% in peripheral blood TCR alpha beta+ cells of normal donors. Here we report two unusual cases, found among 100 healthy individuals studied, who showed an abnormally elevated frequency of these T cells, ie, 5% to 10% and 14% to 19%. Southern blot analyses of the TCR alpha beta+CD4–8- clones all showed the identical rearrangement patterns for each individual, demonstrating that these are derivatives of a single T cell. The same rearrangement patterns were also observed for the freshly isolated lymphocytes of TCR alpha beta+CD4-CD8- fraction, which excludes the possible bias in the processes of in vitro cloning. These TCR alpha beta+CD4–8- T cells were found to express other mature T-cell markers such as CD2, CD3, and CD5 antigens, as well as natural killer (NK) cell markers (CD11b, CD16, CD56, and CD57 antigens) for both individuals. Further, although lectin-dependent or redirected antibody- dependent cell-mediated cytotoxicities were observed for both freshly sorted lymphocytes of TCR alpha beta+CD4–8- fraction and in vitro established clones, NK-like activity was not detected.

Download Full-text