cAMP inhibits both Ras and Rap1 activation in primary human T lymphocytes, but only Ras inhibition correlates with blockade of cell cycle progression

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 998-1006 ◽  
Author(s):  
Thomas Grader-Beck ◽  
Andre A. F. L. van Puijenbroek ◽  
Lee M. Nadler ◽  
Vassiliki A. Boussiotis
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
T Lymphocytes ◽  
T Cell Activation ◽  
Phorbol Ester ◽  
Cell Cycle Progression ◽  
Cell Activation ◽  
Cytokine Production ◽  
Cycle Progression ◽  
Rap1 Activation

Abstract Cyclic adenosine monophosphate (cAMP) is a negative regulator of T-cell activation. However, the effects of cAMP on signaling pathways that regulate cytokine production and cell cycle progression remain unclear. Here, using primary human T lymphocytes in which endogenous cAMP was increased by the use of forskolin and 3-isobutyl-1-methylxanthine (IBMX), we show that increase of cAMP resulted in inhibition of T-cell receptor (TCR)/CD3 plus CD28–mediated T-cell activation and cytokine production and blockade of cell cycle progression at the G1 phase. Increase of cAMP inhibited Ras activation and phosphorylation of mitogen-induced extracellular kinase (MEK) downstream targets extracellular signal–related kinase 1/2 (ERK1/2) and phosphatidylinositol-3-kinase (PI3K) downstream target protein kinase B (PKB; c-Akt). These functional and biochemical events were secondary to the impaired activation of ZAP-70 and phosphorylation of LAT and did not occur when cells were stimulated with phorbol ester, which bypasses the TCR proximal signaling events and activates Ras. Increase of cAMP also inhibited activation of Rap1 mediated by TCR/CD3 plus CD28. Importantly, inhibition of Rap1 activation by cAMP was also observed when cells were stimulated with phorbol ester, although under these conditions Ras was activated and cells progressed into the cell cycle. Thus, TCR plus CD28–mediated activation of ERK1/2 and PKB, cytokine production, and cell cycle progression, all of which are inhibited by cAMP, require activation of Ras but not Rap1. These results indicate that signals that regulate cAMP levels after encounter of T cells by antigen will likely determine the functional fate toward clonal expansion or repression of primary T-cell responses.

scholarly journals CTLA-4 ligation blocks CD28-dependent T cell activation.

1996 ◽  
Vol 183 (6) ◽  
pp. 2541-2550 ◽  
Author(s):  
T L Walunas ◽  
C Y Bakker ◽  
J A Bluestone
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Survival ◽  
T Cell Activation ◽  
Cell Cycle Progression ◽  
Cell Activation ◽  
Cell Function ◽  
Receptor Expression ◽  
Negative Regulator ◽  
Cycle Progression

CTLA-4 is a CD28 homologue believed to be a negative regulator of T cell function. However, the mechanism of this downregulatory activity is not well understood. The present study was designed to examine the effect of CTLA-4 ligation on cytokine production, cell survival, and cell cycle progression. The results demonstrate that the primary effect of CTLA-4 ligation is not the induction of apoptosis. Instead, CTLA-4 signaling blocks IL-2 production, IL-2 receptor expression, and cell cycle progression of activated T cells. Moreover, the effect of CTLA-4 signaling was manifested after initial T cell activation. Inhibition of IL-2 receptor expression and cell cycle progression was more pronounced at late (72 h) time points after initial activation. The effects of anti-CTLA-4 mAbs were most apparent in the presence of optimal CD28-mediated costimulation consistent with the finding that CTLA-4 upregulation was CD28-dependent. Finally, the addition of exogenous IL-2 to the cultures restored IL-2 receptor expression and T cell proliferation. These results suggest that CTLA-4 signaling does not regulate cell survival or responsiveness to IL-2, but does inhibit CD28-dependent IL-2 production.

Imatinib Mesylate Disrupts Cell Cycle Progression, Modifies the Nucleoskeleton and Suppresses Activation-Induced Transcription in Human T Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2914-2914
Author(s):  
Allan Dietz ◽  
William B. Johnson ◽  
Gaylord J. Knutson ◽  
Peggy A. Bulur ◽  
Bertie Schulenberg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Imatinib Mesylate ◽  
T Cell Activation ◽  
Cell Cycle Progression ◽  
Cell Activation ◽  
Structural Changes ◽  
Activated T Cells ◽  
Cycle Progression

Abstract Imatinib mesylate (imatinib, Gleevec®, Novartis, Basel, Switzerland) inhibits T cells in vitro and in vivo (Dietz et al., Blood104: 1094–1099, 2004; Cwynarski et al., Leukemia18: 1332–1339, 2004). The drug blocks T cell cycle progression rather uniquely as it neither inhibits expression of CD69, an early marker of T cell activation, nor induces apoptosis. To characterize the molecular effects of imatinib leading to this mode of T-cell inhibition, we measured the changes in transcriptome (by Affymetrix U133 chips), proteome and phosphoproteome (by Western blotting, differential phosphoprotein expression and mass spectrometry). We found that phytohemagglutinin activated T cells pre-treated with imatinib had reduced expression of 983 transcripts and increased expression of 271 transcripts when compared to untreated PHA activated T cells by the factor of 1.5 or more (p<0.05). Among the prominently down-regulated transcripts were granzyme B, CTLA-4 and IL-2-receptor α-chain (CD25), all characteristic of activated T cells, as well as cyclins D2 and D3 and cyclin-dependent kinases 3, 4 and 7, the molecules regulating cell cycle progression. Among the up-regulated transcripts were Kruppel-like transcription factors 2 and 7, and p27, a finding compatible with the observed cell cycle inhibition. Furthermore, we selected and identified 30 proteins from 2-D gels that were up-regulated and/or hyperphosphorylated in imatinib treated activated T cells. Among these were four heterogeneous ribonucleoproteins, three lamins and γ-actin, all components of the nucleoskeleton at the interface of chromatin and inner nuclear membrane and involved in replication and transcription (Herrmann and Foisner, Cell. Mol. Life Sci.60: 1607–1612, 2003; Shumaker et al. Curr. Opinion Cell Biol.15: 358–366, 2003). Thus, imatinib-borne interference with T cell signal transduction affects the nuclear structure indicating for the first time that nucleoskeleton structural changes are associated with T cell activation status.

scholarly journals CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells.

1996 ◽  
Vol 183 (6) ◽  
pp. 2533-2540 ◽  
Author(s):  
M F Krummel ◽  
J P Allison
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Cycle Progression ◽  
Cell Activation ◽  
Apoptotic Cell ◽  
Activated T Cells ◽  
Activated State

While interactions between CD28 and members of the B7 family costimulate and enhance T cell responses, recent evidence indicates that the CD28 homologue CTLA-4 plays a downregulatory role. The mechanism by which this occurs is not clear, but it has been suggested that CTLA-4 terminates ongoing responses of activated T cells, perhaps by induction of apoptosis. Here we demonstrate that CTLA-4 engagement by antibody cross-linking or binding to B7 inhibits proliferation and accumulation of the primary T cell growth factor, IL-2, by cells stimulated with anti-CD3 and anti-CD28. This inhibition is not a result of enhanced cell death. Rather it appears to result from restriction of transition from the G1 to the S phase of the cell cycle. Our observation that upregulation of both the IL-2R alpha chain and the CD69 activation antigen are inhibited by CTLA-4 engagement supplies further evidence that CTLA-4 restricts the progression of T cells to an activated state. Together this data demonstrates that CTLA-4 can regulate T cell activation in the absence of induction of apoptotic cell death.

scholarly journals p70s6k Integrates Phosphatidylinositol 3-Kinase and Rapamycin-Regulated Signals for E2F Regulation in T Lymphocytes

1999 ◽  
Vol 19 (7) ◽  
pp. 4729-4738 ◽  
Author(s):  
Paul Brennan ◽  
J. W. Babbage ◽  
G. Thomas ◽  
Doreen Cantrell
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Cycle Progression ◽  
Transcriptional Activity ◽  
Interleukin 2 ◽  
Cell Cycle Checkpoint ◽  
Phosphatidylinositol 3 Kinase ◽  
Cycle Progression ◽  
Phosphatidylinositol 3

ABSTRACT In T lymphocytes, the hematopoietic cytokine interleukin-2 (IL-2) uses phosphatidylinositol 3-kinase (PI 3-kinase)-induced signaling pathways to regulate E2F transcriptional activity, a critical cell cycle checkpoint. PI 3-kinase also regulates the activity of p70s6k, the 40S ribosomal protein S6 kinase, a response that is abrogated by the macrolide rapamycin. This immunosuppressive drug is known to prevent T-cell proliferation, but the precise point at which rapamycin regulates T-cell cycle progression has yet to be elucidated. Moreover, the effects of rapamycin on, and the role of p70s6k in, IL-2 and PI 3-kinase activation of E2Fs have not been characterized. Our present results show that IL-2- and PI 3-kinase-induced pathways for the regulation of E2F transcriptional activity include both rapamycin-resistant and rapamycin-sensitive components. Expression of a rapamycin-resistant mutant of p70s6k in T cells could restore rapamycin-suppressed E2F responses. Thus, the rapamycin-controlled processes involved in E2F regulation appear to be mediated by p70s6k. However, the rapamycin-resistant p70s6k could not rescue rapamycin inhibition of T-cell cycle entry, consistent with the involvement of additional, rapamycin-sensitive pathways in the control of T-cell cycle progression. The present results thus show that p70s6k is able to regulate E2F transcriptional activity and provide direct evidence for the first time for a link between IL-2 receptors, PI 3-kinase, and p70s6k that regulates a crucial G1 checkpoint in T lymphocytes.

Pharmacologic Inhibition of Src Family Kinases Differentially Affects T Cell Cytokine Production and Proliferation: Implications for Novel Immunomodulatory Therapies.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1349-1349
Author(s):  
Andrew E. Schade ◽  
Anna M. Jankowska ◽  
Hadrian Szpurka ◽  
Jaroslaw P. Maciejewski
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Production ◽  
Akt Pathway ◽  
T Cell Proliferation ◽  
Tcr Signaling ◽  
Targeted Inhibition

Abstract It is well-recognized that the T cell receptor (TCR) signaling pathway, catalyzed by the Src family kinase (SFK) Lck, is the essential first step in T cell immune responses. The long-held goal of specifically targeting SFK activity for immunomodulatory therapy is becoming more of a reality as SFK inhibitors are entering into clinical use. In particular, dasatinib is an oral small molecule inhibitor of Abl and SFK, including Lck. Given the central importance of Lck in transmitting signals from the TCR signaling complex, and the potent ability of dasatinib to inhibit Lck activity, we hypothesized this agent could provide a novel route of immunomodulation via targeted inhibition of antigen-induced signaling, particularly in combination with currently available immunomodulatory agents. Using intracellular phospho-flow cytometry and western blotting, we show that low nanomolar concentrations of dasatinib potently inhibit TCR induced global protein tyrosine phosphoryation, including the key signaling elements CD3, ZAP-70, LAT, and PLC. In addition, the second messenger pathway Ras/Raf/MEK/ERK is potently blocked, whereas the PI3K-AKT pathway is minimally affected. Furthermore, induction of many pro-inflammatory cytokines, including TNF-α, IFN-γ, IL-2, GM-CSF, IL-17, IP-10, RANTES, and IL-6 (measured by cytokine antibody array) was significantly inhibited when the TCR was triggered in the presence of dasatinib. T cell proliferation, assessed by flow cytometric CFSE dilution analysis, was inhibited in a dose-dependent manner with dasatinib, showing complete inhibtion at 10 nM. Cell cycle analysis based on DNA content by PI staining revealed that dasatinib-treated T cells failed to enter the cell cycle. Considering that the PI3K-AKT pathway was only minimally affected by dasatinib, we examined the ability to achieve enhanced inhibition by using sub-optimal doses of dasatinib and rapamycin that individually produce mild-moderate inhibition of proliferation (20% and 40% inhibition, respectively). When dasatinib and rapamycin are combined at these lower doses, there was a consistently enhanced inhibitory effect on T cell proliferation (80–95% inhibition). Concurrent CD28 stimulation failed to overcome the inhibitory effects of SFK inhibition when the inhibitor was present throughout the stimulation. Interestingly, when SFK inhibition was delayed for at least 24 hours after the initiation of T cell activation with concurrent CD28 stimulation, we observed a divergent effect with regard to cytokine production and proliferation. Pro-inflammatory cytokine production was significantly decreased even if SFK activity was inhibited 48 hours after stimulating T cells, suggesting that ongoing TCR-dependent SFK activity is essential for cytokine production. However, late inhibition of SFK activity with dasatinib increased proliferation, with greater numbers of T cells achieving more rounds of cell division. Thus, while necessary for cytokine production, once the cells have entered the cell cycle, SFK activity regulates negative feedback in CD28-mediated proliferation. In conclusion, targeted inhibition of SFK activity is a promising approach for novel immunomodulatory therapy, particularly in combination with other signaling pathway inhibitors, but timing of SFK inhibition in relation to T cell activation could have an important impact on the immunomodulatory effect.

613 HER2-XPAT, a novel protease-activatable prodrug T cell engager (TCE), with potent T-cell activation and efficacy in solid tumors and large predicted safety margins in non-human primate (NHP)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A649-A649
Author(s):  
Fiore Cattaruzza ◽  
Ayesha Nazeer ◽  
Zachary Lange ◽  
Caitlin Koski ◽  
Mikhail Hammond ◽  
...  
Keyword(s):  
T Cell ◽  
Solid Tumors ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cytokine Production ◽  
Antigenic Specificity ◽  
The Core ◽  
Protease Cleavage ◽  
Safety Margins

BackgroundTCEs are effective in leukemias but have been challenging in solid tumors due to on-target, off-tumor toxicity. Attempts to circumvent CRS include step-up dosing and/or complex designs but are unsuccessful due to toxicity and/or enhanced immunogenicity. HER2-XPAT, or XTENylated Protease-Activated bispecific T-Cell Engager, is a prodrug TCE that exploits the protease activity present in tumors vs. healthy tissue to expand the therapeutic index (TI). The core of the HER2-XPAT (PAT) consists of 2 tandem scFvs targeting CD3 and HER2. Attached to the core, two unstructured polypeptide masks (XTEN) sterically reduce target engagement and extend T1/2. Protease cleavage sites at the base of the XTEN masks enable proteolytic activation of XPATs in the tumor microenvironment, unleashing a potent TCE with short T1/2, further improving the TI. HER2-XPAT, a tumor protease-activatable prodrug with wide safety margins, can co-opt T-cells regardless of antigenic specificity to induce T-cell killing of HER2+ tumors.MethodsPreclinical studies were conducted to characterize the activity of HER2-XPAT, HER2-PAT (cleaved XPAT), and HER2-NonClv (a non-cleavable XPAT) for cytotoxicity in vitro, for anti-tumor efficacy in xenograft models, and for safety in NHPs.ResultsHER2-PAT demonstrated potent in vitro T-cell cytotoxicity (EC50 1-2pM) and target-dependent T-cell activation and cytokine production by hPBMCs. HER2-XPAT provided up to 14,000-fold protection against killing of HER2 tumor cells and no cytotoxicity against cardiomyocytes up to 1uM. In vivo, HER2-XPAT induced complete tumor regressions in BT-474 tumors with equimolar dosing to HER2-PAT, whereas HER2-NonClv had no efficacy, supporting requirement of protease cleavage for T-cell activity. In NHP, HER2-XPAT has been dose-escalated safely up to 42mg/kg (MTD). HER2-XPAT demonstrated early T-cell margination at 2 mg/kg but largely spared CRS, cytokine production, and tissue toxicity up to 42 mg/kg. PK profiles of HER2-XPAT and HER2-NonClv were comparable, consistent with ex vivo stability for cleavage when incubated in cancer pts plasma for 7 days at 37°C. HER2-PAT by continuous infusion induced lethal CRS and cytokine spikes at 0.3 mg/kg/d but was tolerated at 0.25 mg/kg/d, providing HER2-XPAT with >1300-fold protection in tolerability vs. HER2-PAT, >4 logs over cytotoxicity EC50s for HER2 cell lines, and a 20-fold safety margin over the dose required for pharmacodynamic activity.ConclusionsHER2-XPAT is a potent prodrug TCE with no CRS and a wide TI based on NHPs. With XTEN’s clinical data demonstrating low immunogenicity, the XPATs are a promising solution. IND studies are ongoing. Additional PK/PD, cytokines, safety, and efficacy data will be presented.

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