Telomerase Activity and Telomere Length of CD4+CD25+ Regulatory T-Cells under Conditions of In Vitro and In Vivo Expansion.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3857-3857
Author(s):  
Dominik G.F. Wolf ◽  
Anna M. Wolf ◽  
Christian Koppelstaetter ◽  
Holger F. Rumpold ◽  
Gert Mayer ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Telomere Length ◽  
Cancer Patients ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Healthy Human ◽  
Telomere Shortening

Abstract The expandability of CD4+CD25+ regulatory T-cells (Treg) has been shown in vitro and in vivo. Activation of telomerase activity is a prerequisite for clonal expansion and telomere maintenance in T-cells. There is currently no data available on the expression and function of telomerase in proliferating Treg. Analyses of telomere length by flow-FISH, real-time PCR and Southern blotting revealed that Treg isolated from healthy human volunteers have significantly shortened telomeres when compared to CD4+CD25− T-cells. However, telomere length is not further shortened in Treg isolated from the peripheral blood of cancer patients, despite the observation that the regulatory T-cell pool of these patients was significantly enlarged. To gain further insight into maintenance of telomere length of Treg, we induced in vitro proliferation of Treg by stimulation with anti-CD3 and IL-2. This led to a rapid increase of telomerase activity, as determined by PCR-ELISA. However, when we focused on the proliferating fraction of Treg using a sorting strategy based on the dilution of CFSE, we could show a significant telomere shortening in Treg with high proliferative and immmuno-suppressive capacity. Of note, proliferating CFSElow Treg are characterized by high telomerase activity, which however seems to be insufficient to avoid further telomere shortening under conditions of strong in vitro stimulation. In contrast, under conditions of in vivo expansion of Treg in cancer patients, the induction of telomerase activity is likely to compensate for further telomere erosion. These data might be of importance when considering the application of in vitro expanded Treg for the treatment of GvHD or autoimmune diseases, as telomere shortening might be associated with genomic instability.

Maintenance of Telomere Length in Peripheral Blood CD4+CD25+ Regulatory T-Cells of Cancer Patients Despite Active Proliferation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3309-3309
Author(s):  
Dominik Wolf ◽  
Holger Rumpold ◽  
Christian Koppelstaetter ◽  
Guenther Gastl ◽  
Eberhard Gunsilius ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Telomere Length ◽  
Cancer Patients ◽  
Peripheral Blood ◽  
Telomerase Activity ◽  
Telomere Shortening ◽  
Active Proliferation

Abstract CD4+CD25+ regulatory T-cells (Treg) are increased in the peripheral blood of cancer patients. It remains unclear whether this is due to redistribution or active proliferation. The latter would require the up-regulation of telomerase activity, whose regulation also remains unknown for Treg. We therefore isolated Treg and the respective CD4+CD25− control T-cell population from peripheral blood of cancer patients (n=23) and healthy age-matched controls (n=17). Analysis of their content of T-cell receptor excision circles (TREC) revealed that the observed increase of Treg frequencies in peripheral blood is due to active cycling rather than to redistribution from other compartments (i.e. secondary lymphoid organs or bone-marrow), as Treg from cancer patients are characterized by a significant decrease of TREC content when compared to TREC content of Treg isolated from healthy age-matched controls. Surprisingly, despite their proven in vivo proliferation, telomere length is not further shortened in Treg from peripheral blood of cancer patients as shown by Flow-Fish, Real-Time PCR and Southern Blotting. Accodingly, telomerase activity of Treg was readily inducible in vitro by OKT3 together with IL-2. Notably, sorting of in vitro proliferating Treg using dilution of CFSE revealed a significant telomere shortening in Treg with high proliferative capacity (i.e. CFSElow fraction) under conditions of strong in vitro stimulatory growth conditions despite a high telomerase activity. Thus, under conditions of strong in vitro stimulation induction of telomerase seems to be insufficient to avoid progressive telomere shortening. In contrast, in actively proliferating peripheral blood Treg from patients with epithelial malignancies induction of telomerase activity is likely to compensate for further telomere erosion.

Importance of T-Cell Proliferative Stress In Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2939-2939
Author(s):  
Lili Yang ◽  
Amber Schmidt ◽  
Rami Komrokji ◽  
Jeffrey S. Painter ◽  
Ashley Cole ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Myelodysplastic Syndrome ◽  
Copy Number ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Htert Mrna ◽  
Cell Compartment

Abstract Abstract 2939 During thymic development, T-cells are produced with a large array of genetically diverse aβ T-cell receptors (TCRs) primed for robust antigen recognition. In patients with myelodysplastic syndrome (MDS), the functional T-cell repertoire becomes contracted and the CD4/CD8 ratio reduced possibly reflecting antigen stimulation. Dynamics within the T-cell compartment are regulated by progressive telomere shortening that occurs with proliferation. Preservation of telomere length is mediated by the telomerase (hTERT) enzyme. In MDS, we previously showed that telomere length in peripheral blood mononuclear cells (PBMCs) was significantly reduced among cases compared to controls. We hypothesized that dysregulated telomere maintenance due to proliferative stress contributes to contraction of the T-cell compartment. First, we examined telomerase mRNA expression and telomerase activity in purified T-cells in a cohort of 32 healthy individuals. The age of these donors ranged from 17 to 82 years with a mean age of 58 years. CD3+ T-cells were separated from the peripheral blood by negative selection (Miltenyi Biotech Corp) and measurements were made in both unstimulated and stimulated cells or 3 days with CD3/CD28 T-cell expanders (Dynabeads®) to induce proliferation. hTERT expression was measured by quantitative RT-PCR (qRT-PCR) and telomerase enzymatic function was quantified by the Telomere Repeat Amplification Protocol (TRAP assay). We found that hTERT mRNA was low- to undetectable in unstimulated T-cells, but was significantly up-regulated by stimulation (1.71±1.45 vs 3.43±1.90, p<0.001). In stimulated T-cells, the amount of inducible hTERT transcription was inversely correlated with age (p=0.0086). Results of telomerase functional assays showed that the stimulated T-cells had dramatically increased telomerase activity compared with unstimulated cells (623.03±435.76 vs 3.71±6.37, p<0.001), but in contrast to hTERT, telomerase enzyme function did not changed with age (p=0.7). Individual purification and stimulation of CD4+ and CD8+ T-cells showed that CD4+ cells induced 4-fold greater increase in telomerase activity compared to CD8+ cells suggesting that the primary requirement for telomeric repair lies with highly proliferative CD4+ T-cells. Next, we measured DNA copy number of T-cell receptor excision circles (TRECs) in both unstimulated and stimulated T-cells as a function of age to determine the capacity of these cells to undergo proliferation in vitro. TRECs are episomal DNA fragments that do not transfer to daughter cells and are expressed exclusively in mature T-cells. After in vitro stimulation, TREC copy number decreased from a mean of 218 to 67 copies indicating that 2.9 population doublings (PD) had occurred within the 3-day incubation period. The number of PDs was maintained with age (p=0.26) suggesting that the proliferation capacity along with the telomerase function normally remains intact with age progression. These results varified the utility of applying TREC analysis to determine the proliferative history in vitro and in vivo. The proliferative history of peripheral T-cells in vivo was then determined in a separate cohort of healthy controls (n=63) age-matched to MDS patients (n=66) by comparing the change in the amount of TREC DNA in unstimulated cells. For every year increase in age, the log TREC values decreased by 0.05 DNA copies (p=0.0012) in healthy donors. In MDS patients, a 2-fold greater decline in TREC copy number occurred each year (0.099 unit decrease in TREC copies per year among cases, p<0.0001; 0.05 copies per year among controls, p=0.012) suggesting that T-cells in MDS patients have been exposed to more proliferative stress. Collectively, our results indicate that telomere maintenance is controlled normally in proliferating T-cells by inducing hTERT mRNA expression and by upregulating telomerase enzyme activity. In MDS cases, accelerated proliferative stress may contribute to repertoire contraction and CD4/CD8 ratio abnormalities. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Dyskerin Is a Component of the Arabidopsis Telomerase RNP Required for Telomere Maintenance

2008 ◽  
Vol 28 (7) ◽  
pp. 2332-2341 ◽  
Author(s):  
Kalpana Kannan ◽  
Andrew D. L. Nelson ◽  
Dorothy E. Shippen
Keyword(s):  
Telomere Length ◽  
Bone Marrow Failure ◽  
Telomerase Activity ◽  
Dyskeratosis Congenita ◽  
Dominant Negative ◽  
Telomere Maintenance ◽  
Dependent Manner ◽  
Missense Mutations

ABSTRACT Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.

Telomere length of in vivo expanded CD4+CD25+ regulatory T-cells is preserved in cancer patients

2005 ◽  
Vol 55 (10) ◽  
pp. 1198-1208 ◽  
Author(s):  
Dominik Wolf ◽  
Holger Rumpold ◽  
Christian Koppelstätter ◽  
Guenther A. Gastl ◽  
Michael Steurer ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Telomere Length ◽  
Cancer Patients

scholarly journals Swc4 positively regulates telomere length independently of its roles in NuA4 and SWR1 complexes

2020 ◽  
Vol 48 (22) ◽  
pp. 12792-12803
Author(s):  
Jia-Cheng Liu ◽  
Qian-Jin Li ◽  
Ming-Hong He ◽  
Can Hu ◽  
Pengfei Dai ◽  
...  
Keyword(s):  
Telomere Length ◽  
Essential Gene ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Direct Role ◽  
Telomere Shortening ◽  
Length Regulation ◽  
Telomere Length Regulation

Abstract Telomeres at the ends of eukaryotic chromosomes are essential for genome integrality and stability. In order to identify genes that sustain telomere maintenance independently of telomerase recruitment, we have exploited the phenotype of over-long telomeres in the cells that express Cdc13-Est2 fusion protein, and examined 195 strains, in which individual non-essential gene deletion causes telomere shortening. We have identified 24 genes whose deletion results in dramatic failure of Cdc13-Est2 function, including those encoding components of telomerase, Yku, KEOPS and NMD complexes, as well as quite a few whose functions are not obvious in telomerase activity regulation. We have characterized Swc4, a shared subunit of histone acetyltransferase NuA4 and chromatin remodeling SWR1 (SWR1-C) complexes, in telomere length regulation. Deletion of SWC4, but not other non-essential subunits of either NuA4 or SWR1-C, causes significant telomere shortening. Consistently, simultaneous disassembly of NuA4 and SWR1-C does not affect telomere length. Interestingly, inactivation of Swc4 in telomerase null cells accelerates both telomere shortening and senescence rates. Swc4 associates with telomeric DNA in vivo, suggesting a direct role of Swc4 at telomeres. Taken together, our work reveals a distinct role of Swc4 in telomere length regulation, separable from its canonical roles in both NuA4 and SWR1-C.

scholarly journals P01.02 TLR-mediated suppression of the CCL22-CCR4 axis as a new target for tumor immunotherapy

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A3.2-A4
Author(s):  
J Grün ◽  
I Piseddu ◽  
C Perleberg ◽  
N Röhrle ◽  
S Endres ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Regulatory T Cells ◽  
Type I ◽  
List Type ◽  
Gm Csf

BackgroundUnmethylated CpG-DNA is a potent ligand for the endosomal Toll-like-receptor-9, important for the immune activation to pathogen-associated molecules.1 CpG and other TLR-ligands show effective immunotherapeutic capacities in cancer treatment by inducing an antitumorigenic immunity.2 They are able to reduce tumor progression by reduction of intratumoral secretion of the immunoregulating chemokine CCL223 and subsequent recruitment of immunosuppressive regulatory T cells (Treg), which express CCR4 the only so far known receptor for CCL22.4 Our recent work has shown that CCL22 secretion by dendritic cells (DC) in the lymph node, mediates tolerance by inducing DC-Treg contacts.5 Indeed, in the absence of CCL22, immune responses to vaccination were stronger and resulted in tumor rejection.6 Therefore, we are aiming to investigate the effects of TLR-ligands on systemic CCL22 levels, elucidating all involved mechanisms to identify new targets for cancer immunotherapy.Materials and MethodsT, B and CD11c+ DCs of wildtype (wt) and RAG1-/- mice were isolated from splenocytes by magnetic-activated cell sorting for in vitro assays. Different co-cultures were incubated with CpG and GM-CSF, known as an CCL22 inducer.5 For in vivo experiments, wt mice were treated with CpG, R484 or poly(I:C) alone and in combination with GM-CSF. CCL22-levels in a number of organs were analyzed.ResultsAnalyzing the different immune cell compartments in vitro, we found that DCs in whole splenocytes secrete CCL22 during culture while DC cultured alone showed no CCL22 secretion. When treated with CpG, CCL22-levels were reduced in splenocytes, while it was induced in DC culture alone. The same results were seen when RAG splenocytes, that lack functional B and T cells, were cultured with CpG. CpG treated B cells were able to suppress CCL22 secretion by DC unlike T cells alone. Co-cultures of T and B cells treated with CpG, however, induced the strongest CCL22 suppression in DC. In vivo, we could show that all TLR ligands tested reduced CCL22 in a number of organs significantly. Furthermore, CpG showed the strongest suppression of CCL22 even in the presence of the CCL22 inducer GM-CSF.5ConclusionsWe could show that B cells with T cells mediate CCL22 suppression by TLR ligands. The fact that CpG was able to reduce CCL22 levels even in the presence of the inducer GM-CSF demonstrates the potent CCL22 suppressive capacity of TLR ligands.ReferencesO’Neill LA, et al. The history of toll-like receptors – redefining innate immunity. Nat Rev Immunol 2013;13(6):453–60.Rothenfusser S, et al. Recent advances in immunostimulatory CpG oligonucleotides. Curr Opin Mol Ther 2003;5(2):98–106.Wang S, et al. Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A 2016;113(46): E7240–E7249.Rapp M, et al. CCL22 controls immunity by promoting regulatory T cell communication with dendritic cells in lymph nodes. J Exp Med 2019;216(5):1170–1181.Piseddu I, et al. Constitutive expression of CCL22 is mediated by T cell-derived GM-CSF. J Immunol 2020;205(8):2056–2065.Anz D, et al. Suppression of intratumoral CCL22 by type i interferon inhibits migration of regulatory T cells and blocks cancer progression. Cancer Res 2015;75(21):4483–93.Disclosure InformationJ. Grün: None. I. Piseddu: None. C. Perleberg: None. N. Röhrle: None. S. Endres: None. D. Anz: None.

scholarly journals Precision engineering of an anti-HLA-A2 chimeric antigen receptor in regulatory T cells for transplant immune tolerance

2021 ◽  
Author(s):  
Yannick D. Muller ◽  
Leonardo M.R. Ferreira ◽  
Emilie Ronin ◽  
Patrick Ho ◽  
Vinh Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Chimeric Antigen Receptor ◽  
Mononuclear Cells ◽  
Antigen Receptor ◽  
Single Chain ◽  
Transplant Tolerance ◽  
Peripheral Blood Mononuclear

Infusion of regulatory T cells (Tregs) engineered with a chimeric antigen receptor (CAR) targeting donor-derived human leukocyte antigen (HLA) is a promising strategy to promote transplant tolerance. Here, we describe an anti-HLA-A2 CAR (A2-CAR) generated by grafting the complementarity-determining regions (CDRs) of a human monoclonal anti-HLA-A2 antibody into the framework regions of the Herceptin 4D5 single-chain variable fragment and fusing it with a CD28-zeta signaling domain. The CDR-grafted A2-CAR maintained the specificity of the original antibody. We then generated HLA-A2 mono-specific human CAR Tregs either by deleting the endogenous T-cell receptor (TCR) via CRISPR/Cas9 and introducing the A2-CAR using lentiviral transduction or by directly integrating the CAR construct into the TCR alpha constant locus using homology-directed repair. These A2-CAR+TCRdeficient human Tregs maintained both Treg phenotype and function in vitro. Moreover, they selectively accumulated in HLA-A2-expressing islets transplanted from either HLA-A2 transgenic mice or deceased human donors. A2-CAR+TCRdeficient Tregs did not impair the function of these HLA-A2+ islets, whereas similarly engineered A2-CAR+TCRdeficientCD4+ conventional T cells rejected the islets in less than 2 weeks. A2-CAR+TCRdeficient Tregs delayed graft-versus-host disease only in the presence of HLA-A2, expressed either by co-transferred peripheral blood mononuclear cells or by the recipient mice. Altogether, we demonstrate that genome-engineered mono-antigen-specific A2-CAR Tregs localize to HLA-A2-expressing grafts and exhibit antigen-dependent in vivo suppression, independent of TCR expression. These approaches may be applied towards developing precision Treg cell therapies for transplant tolerance.

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