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

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.

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A novel myelin P0–specific T cell receptor transgenic mouse develops a fulminant autoimmune peripheral neuropathy

Journal of Experimental Medicine ◽

10.1084/jem.20082113 ◽

2009 ◽

Vol 206 (3) ◽

pp. 507-514 ◽

Cited By ~ 31

Author(s):

Cédric Louvet ◽

Beniwende G. Kabre ◽

Dan W. Davini ◽

Nicolas Martinier ◽

Maureen A. Su ◽

...

Keyword(s):

T Cells ◽

Peripheral Neuropathy ◽

T Cell ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Chronic Inflammatory Demyelinating Polyneuropathy ◽

Gene Knockout ◽

Premature Death ◽

Cell Receptor

Autoimmune-prone nonobese diabetic mice deficient for B7-2 spontaneously develop an autoimmune peripheral neuropathy mediated by inflammatory CD4+ T cells that is reminiscent of Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy. To determine the etiology of this disease, CD4+ T cell hybridomas were generated from inflamed tissue–derived CD4+ T cells. A majority of T cell hybridomas were specific for myelin protein 0 (P0), which was the principal target of autoantibody responses targeting nerve proteins. To determine whether P0-specific T cell responses were sufficient to mediate disease, we generated a novel myelin P0–specific T cell receptor transgenic (POT) mouse. POT T cells were not tolerized or deleted during thymic development and proliferated in response to P0 in vitro. Importantly, when bred onto a recombination activating gene knockout background, POT mice developed a fulminant form of peripheral neuropathy that affected all mice by weaning age and led to their premature death by 3–5 wk of age. This abrupt disease was associated with the production of interferon γ by P0-specific T cells and a lack of CD4+ Foxp3+ regulatory T cells. Collectively, our data suggest that myelin P0 is a major autoantigen in autoimmune peripheral neuropathy.

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The presence of interleukin 4 during in vitro priming determines the lymphokine-producing potential of CD4+ T cells from T cell receptor transgenic mice.

Journal of Experimental Medicine ◽

10.1084/jem.176.4.1091 ◽

1992 ◽

Vol 176 (4) ◽

pp. 1091-1098 ◽

Cited By ~ 671

Author(s):

R A Seder ◽

W E Paul ◽

M M Davis ◽

B Fazekas de St Groth

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Cell Receptor ◽

Inhibitory Effect ◽

Interleukin 4 ◽

Ifn Gamma

To study the factors that determine whether CD4+ T cells produce interleukin 4 (IL-4) or interferon gamma (IFN-gamma) upon stimulation we used a system allowing naive T cells to be primed in vitro by specific antigen. Dense CD4+ T cells were purified from mice that expressed transgenes encoding a T cell receptor specific for pigeon cytochrome C peptide 88-104 in association with I-Ek. These T cells produced very limited amounts of IL-4 and IFN-gamma upon immediate challenge with 88-104 and antigen-presenting cells (APC). However, after an initial "priming" culture in which they were incubated for 4 d in the presence of 88-104, APC, and 1,000 U/ml IL-4, the T cells acquired the capacity to produce substantial amounts of IL-4 upon rechallenge but made very little IFN-gamma. Cells primed in the absence of IL-4 produced IFN-gamma upon rechallenge but virtually no IL-4. The inhibitory effect of IL-4 on IFN-gamma production did not appear to be mediated by the induction of IL-10 production since IL-10 addition to initial cultures did not suppress priming for IFN-gamma production, nor did anti-IL-10 block the inhibitory effect of IL-4. IFN-gamma itself did not increase priming for IFN-gamma production, nor did anti-IFN-gamma reduce such priming. IFN-gamma did, however, diminish priming for IL-4 production when limiting amounts of IL-4 (100 U/ml) were used in the initial culture. The dominant effect of IL-4 in determining the lymphokine-producing phenotype of primed cells was observed with dendritic cells (DC), activated B cells, and I-Ek-transfected fibroblasts as APC. However, the different APC did vary in their potency, with DC being superior to activated B cells, which were superior to transfected fibroblasts.

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Transient Inhibition of Interleukin 4 Signaling by T Cell Receptor Ligation

Journal of Experimental Medicine ◽

10.1084/jem.192.8.1125 ◽

2000 ◽

Vol 192 (8) ◽

pp. 1125-1134 ◽

Cited By ~ 48

Author(s):

Jinfang Zhu ◽

Hua Huang ◽

Liying Guo ◽

Timothy Stonehouse ◽

Cynthia J. Watson ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Cell Receptor ◽

Mitogen Activated Protein Kinase ◽

Interleukin 4 ◽

Interferon Α ◽

Limiting Factor

Interleukin (IL)-4 and IL-12 together with T cell receptor (TCR) engagement are crucial for the differentiation of CD4+ T cells into T helper (Th)2 or Th1 cells, respectively. Although IL-4 receptors (IL-4Rs) but not IL-12Rs are expressed on naive CD4+ T cells, IL-4 has no apparent advantage over IL-12 in driving naive T cell differentiation when the cells are primed with both IL-4 and IL-12 in vitro. It was found that IL-4–induced phosphorylation of Janus kinases 1 and 3, IL-4Rα, signal transducer and activator of transcription 6, and insulin receptor substrate 2 was strikingly but transiently inhibited by TCR ligation both in conventional and TCR transgenic T cells. TCR engagement also blocked the expression of an IL-4–inducible gene. Signals induced by other cytokines, including IL-2, IL-6, and interferon α, but not by insulin-like growth factor 1, were also blocked by TCR engagement. The capacity of various inhibitors to reverse TCR-mediated inhibition of IL-4 signaling suggested that activation of the Ras–mitogen-activated protein kinase pathway and of the calcineurin pathway contribute to desensitizing IL-4R. IL-4 responsiveness returned at about the time (∼12 h) that IL-12–mediated signaling was first observed. Thus, through different mechanisms, neither IL-4R nor IL-12R has any clear advantage in polarizing cells; rather, the availability of cytokine is probably the limiting factor in this process.

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Ceramide Synthase 2 Null Mice Are Protected from Ovalbumin-Induced Asthma with Higher T Cell Receptor Signal Strength in CD4+ T Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22052713 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2713

Author(s):

Sun-Hye Shin ◽

Kyung-Ah Cho ◽

Hee-Soo Yoon ◽

So-Yeon Kim ◽

Hee-Yeon Kim ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Acyl Chain ◽

Signal Strength ◽

Cell Receptor ◽

Ceramide Synthase ◽

Asthmatic Patients

(1) Background: six mammalian ceramide synthases (CerS1–6) determine the acyl chain length of sphingolipids (SLs). Although ceramide levels are increased in murine allergic asthma models and in asthmatic patients, the precise role of SLs with specific chain lengths is still unclear. The role of CerS2, which mainly synthesizes C22–C24 ceramides, was investigated in immune responses elicited by airway inflammation using CerS2 null mice. (2) Methods: asthma was induced in wild type (WT) and CerS2 null mice with ovalbumin (OVA), and inflammatory cytokines and CD4 (cluster of differentiation 4)+ T helper (Th) cell profiles were analyzed. We also compared the functional capacity of CD4+ T cells isolated from WT and CerS2 null mice. (3) Results: CerS2 null mice exhibited milder symptoms and lower Th2 responses than WT mice after OVA exposure. CerS2 null CD4+ T cells showed impaired Th2 and increased Th17 responses with concomitant higher T cell receptor (TCR) signal strength after TCR stimulation. Notably, increased Th17 responses of CerS2 null CD4+ T cells appeared only in TCR-mediated, but not in TCR-independent, treatment. (4) Conclusions: altered Th2/Th17 immune response with higher TCR signal strength was observed in CerS2 null CD4+ T cells upon TCR stimulation. CerS2 and very-long chain SLs may be therapeutic targets for Th2-related diseases such as asthma.

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Functional Uncoupling of T-cell Receptor Engagement and Lck Activation in Anergic Human Thymic CD4+T Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m101072200 ◽

2001 ◽

Vol 276 (20) ◽

pp. 17455-17460 ◽

Cited By ~ 12

Author(s):

Wakae Fujimaki ◽

Makio Iwashima ◽

Junji Yagi ◽

Hua Zhang ◽

Hisako Yagi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Cd4 T Cells ◽

Cell Receptor

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T cell receptor zeta/CD3-p59fyn(T)-associated p120/130 binds to the SH2 domain of p59fyn(T).

Journal of Experimental Medicine ◽

10.1084/jem.178.6.2107 ◽

1993 ◽

Vol 178 (6) ◽

pp. 2107-2113 ◽

Cited By ~ 46

Author(s):

A J da Silva ◽

O Janssen ◽

C E Rudd

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

T Cell Activation ◽

Intracellular Signaling ◽

Cell Activation ◽

Cell Receptor ◽

Sh2 Domain ◽

T Complex

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.

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CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway

Blood ◽

10.1182/blood-2009-01-200923 ◽

2009 ◽

Vol 114 (3) ◽

pp. 580-588 ◽

Cited By ~ 45

Author(s):

Kathrin Gollmer ◽

François Asperti-Boursin ◽

Yoshihiko Tanaka ◽

Klaus Okkenhaug ◽

Bart Vanhaesebroeck ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Early Time ◽

Cell Receptor ◽

Tcr Signaling ◽

Activation Markers

Abstract CD4+ T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)–transgenic (tg) CD4+ T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3KδD910A/D910A or PI3Kγ-deficient TCR-tg CD4+ T cells showed similar responsiveness to CCL21 costimulation as control CD4+ T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4+ T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca2+ signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

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T-CELL RECEPTOR INDEPENDENT ACTIVATION OF CD4+T-CELLS IN SEVERE ACUTE PANCREATITIS

Pancreas ◽

10.1097/01.mpa.0000335444.45291.a0 ◽

2008 ◽

Vol 37 (4) ◽

pp. 468

Author(s):

A. Dummer ◽

M. Sendler ◽

F.-U. Weiss ◽

B. M. Bröker ◽

M. M. Lerch ◽

...

Keyword(s):

T Cells ◽

Acute Pancreatitis ◽

T Cell ◽

T Cell Receptor ◽

Severe Acute Pancreatitis ◽

Cd4 T Cells ◽

Cell Receptor

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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.

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