T-Cell Cycle and Immunosenescence: Role of Aging in the T-Cell Proliferative Behavior and Status Quo Maintenance

Abstract The bone marrow (BM) is a complex microsystem to support lifelong blood production. At steady-state most hematopoietic stem cells (HSC) are quiescent. However, in situations of increased demand, their activation is triggered by an array of signals, such as cytokines. Following hematopoietic cell transplantation (HCT) in the phase of hematopoietic reconstitution maximal blood production is needed. In an HCT donor HSC are given together with immune cells in the belief that T cells support HSC engraftment and regeneration of the blood system. Yet, states of immune mediated BM insufficiency, hypoplasia, and cytopenias are often observed. Moreover, with increased use of reduced intensity conditioning (RIC) engraftment failure has reemerged as a serious problem. Here, we studied the effects of distinct T cell subsets on hematopoietic reconstitution following HCT; specifically, we examined how conventional CD4+CD25- T cells (CD4conv) vs regulatory T cells (CD4+CD25+, Treg) modify the BM environment and influence donor-HSC activity and engraftment. We used minor-mismatched mouse models, non-myeloablative total body irradiation (TBI) conditioning and transplantations of purified HSC (KTLS; cKit+Thy1.1loLin–Sca-1+) plus selected T cell subsets. Recipients of HSC, HSC+Treg or HSC+CD8+ - but not HSC+CD4conv- demonstrated prompt donor engraftment with mixed chimerism in all lineages. Transplantation of HSC+Treg resulted in significantly faster lymphocyte recovery and higher levels of donor chimerism compared with recipients of HSC, HSC+CD8+ or HSC+CD4conv. Particularly B-cell regeneration was markedly higher in HSC+Treg-recipients compared with all other groups. In contrast, (B-) lymphopoiesis was severely impaired in recipients of HSC+CD4conv; when lymphocytes recovered eventually they were of host type. Moreover, in BM and spleens of HSC+CD4conv recipients pronounced hypocellularity was observed. This suppression of hematopoiesis was due to IFNg secretion of donor CD4conv cells, which were activated by dendritic cells via IL-12. High cytokine levels (of both IL-12 and IFNg) were only detectable in the BM (and not the spleen) of HSC+CD4conv recipients, where they resulted in an arrest of early hematopoiesis at the stage of short-term HSC and decreased cell-cycle activity within the progenitor compartment. As a consequence more mature multipotent progenitors were lacking. The key role of IFNg in halting hematopoietic maturation was confirmed by using CD4conv cells from IFNg-/- mice, which had no suppressive effects on BM cellularity and maturation of blood cells; rather, recipients of HSC+IFNg-/-CD4conv cells had equivalent cell numbers and subset distributions as mice given HSC alone. We hypothesized that differences of hematopoietic regeneration and donor engraftment relate to cell cycle activity of HSC in presence of CD4conv vs Tregs. To study the influence of these CD4-subsets on HSC cycling in more detail FACS-purified Treg vs CD4conv cells were infused into congenic mice following low-dose TBI-stimulation. In fact, on d+8 post-infusion HSC in the BM of Treg-recipients had increased cell-cycling activity in long-term-HSC and multipotent-progenitor fractions compared with mice given CD4conv cells or radiation only. These data lead us to speculate that Tregs promote, directly or indirectly, HSC proliferation; in the context of an allogeneic HCT this increased cycling of host HSC my open-up the niche space required to allow donor HSC to engraft. In contrast, CD4convinhibited HSC cycling activity, resulting in BM hypoplasia and cytopenias; at the same time donor HSC engraftment was impaired due to HSC-niche occupation by quiescent host HSC. Our findings underscore the critical role of T cells in regulating hematopoiesis under physiologic conditions and even more following allogeneic HCT. While donor T cells are generally believed to improve regeneration of the blood post-HCT and to be required to overcome host barriers, our data suggest Treg facilitate engraftment and hematopoiesis by increasing HSC cycling-activity and thereby making marrow sites available. CD4conv appear to have the opposite effect, resulting in decreased HSC proliferation and maturation - thus occupation of HSC niches. Our studies are of particular relevance to allogeneic HCT settings using RIC, where host HSC persist and grafts can be rejected by residual host immune cells. Disclosures No relevant conflicts of interest to declare.

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Insights Into the Role of ASPP2 (Apoptosis Stimulating Protein of p53-2) in Lymphomagenesis.

Blood ◽

10.1182/blood.v114.22.1967.1967 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1967-1967

Author(s):

Kerstin M Kampa ◽

Sandra Mueller ◽

Michael Bonin ◽

Marcus M Schittenhelm ◽

Charles D Lopez

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

T Cell ◽

Cell Growth ◽

Tumor Suppressor ◽

Target Genes ◽

Tumor Formation ◽

Transcriptional Networks ◽

Damage Response

Abstract Abstract 1967 Poster Board I-990 ASPP2 is a member of a family of p53 binding proteins that enhance apoptosis, in part through selective stimulation of p53 transactivation of pro-apoptotic target genes. Low ASPP2 expression is found in many human cancers and has been associated with poor clinical outcome in patients with aggressive lymphoma. Using an ASPP2+/- mouse model, we have previously demonstrated that ASPP2 is a haploinsufficient tumor suppressor and that reduced ASPP2 expression results in attenuated damage-response thresholds (Kampa et al., PNAS 2009). While ASPP2-/- mice are not viable, ASPP2+/- mice have an increased incidence of -irradiation-induced tumors compared to ASPP2+/+ mice.γspontaneous and ASPP2+/- mice develop high-grade thymic T-cell lymphomas after -irradiation. Moreover, primary ASPP2+/- thymocytes have an attenuatedγ -irradiation compared to ASPP2+/+ thymocytes.γapoptotic response after To explore the mechanisms of how attenuated ASPP2 expression could increase thymic lymphomagenesis and attenuate apoptosis, we performed global gene expression profiling on unirradiated, and 5 Gy irradiated ASPP2+/+ and ASPP2+/- thymocytes using an Affymetrix Mouse GeneChip® Array. We found significant differences in gene expression between ASPP2+/+ and ASPP2+/- thymocytes, in both unirradiated and irradiated sets. Using Ingenuity Pathway Analysis software, we found that amongst the highest scoring pathways displaying differences were those associated with cell growth, tumor formation, hematologic malignancies, immune response, cell death and cell cycle regulation. We additionally studied global phosphorylation patterns using 2-dimensional gel electrophoresis, fluorescent phosphoprotein dye Pro-Q Diamond staining, and liquid chromatography tandem mass spectrometry to determine the posttranscriptional mechanisms mediated by attenuated ASPP2 expression. Analysis of the phosphoproteome of ASPP2+/+ and ASPP2+/- mouse embryonic fibroblasts (with and without irradiation) revealed differences in the phosphorylation status of 108 peptides/proteins including those involved in regulating cell cycle checkpoints, T-cell receptor signaling, cell stress response, DNA repair mechanisms, cell growth, translation and transcription. Differential expression of the identified genes and proteins was verified by PCR and Western Blot. Thus, reduced ASPP2 expression affects global transcriptional as well as post-transcriptional networks intimately involved in the development of hematologic disorders–suggesting that ASPP2 function is by far more complex than solely enhancing the expression of pro-apoptotic p53 target genes. Given that ASPP2 is a bona fide tumor suppressor, reduced ASPP2 levels result in global dysregulation of pathways engaged in tumor suppression networks and the cellular damage response, which may ultimately promote genomic instability and tumor formation. Our findings provide insights into the role of ASPP2 in lymphomagenesis and reveal possible new targets for cancer therapy. Disclosures: No relevant conflicts of interest to declare.

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mTORC1 Inactivation Prevents and Eradicates Acute Lymphoblastic T-Cell Leukemia

Blood ◽

10.1182/blood.v122.21.1211.1211 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1211-1211

Author(s):

Takayuki Hoshii ◽

Atsuo Kasada ◽

Tomoki Hatakeyama ◽

Masashi Ohtani ◽

Yuko Tadokoro ◽

...

Keyword(s):

Cell Cycle ◽

T Cell ◽

T Cell Development ◽

Cell Development ◽

Cyclin D3 ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Rapamycin Treatment ◽

T Cell Progenitors

Abstract mTOR is a serine/threonine kinase that has a central role in the regulation of cell growth and cell metabolism and forms two functionally different complexes, named mTORC1 and mTORC2. Despite the effectiveness of rapamycin, an allosteric mTOR inhibitor, in immunosuppression, the precise roles of mTORCs in T-cell development remain unclear. Here we show that mTORC1 plays a critical role in the earliest development of T-cell progenitors. To understand the physiological role of mTORC1 in T-cell development, we evaluated the effects of mTORC1 inhibition by rapamycin treatment or the genetic deletion of the Raptor gene, an essential component of mTORC1. Raptor deficiency dramatically inhibited the development of CD4/CD8 double-positive (DP) cells. Rapamycin treatment produced similar defects, but to a lesser extent. Deficiency of Raptor, but not Rictor, a mTORC2 component, resulted in abnormality of cell cycle of early T-cell progenitors, associated with instability of the Cyclin D3/CDK6 complex, indicating that mTORC1 and 2 control T-cell development in different manners. When we treated T-cells with a proteasome inhibitor, MG-132, in vitro, the reduction of Cyclin D3 and CDK6 by mTORC1 inactivation was reversed. These data suggest that mTORC1 activity may control the Cyclin D3/CDK6 complex via post-transcriptional mechanisms. In a model of myeloproliferative neoplasm (MPN) and T-cell leukemia (T-ALL) evoked by Kras activation, rapamycin treatment prevents development of T-ALL, but not MPN. After the onset of T-ALL, rapamycin-insensitive Notch-driven T-ALL cells survived in vivo. Raptor deficiency dramatically inhibited proliferation of oncogenic Kras–expressing T-cell progenitors and prevents the development of T-ALL, but not MPN. In contrast to T-cell progenitors, cell cycle of myeloid progenitors was not affected by mTORC1 inactivation. Phosphorylation of p70S6K and 4E-BP1, direct substrates of mTORC1, was apparently decreased in Raptor-deficient myeloid cells. Interestingly, consistent with hypo-phosphorylation of p70S6K and 4E-BP1, rates of newly synthesized protein were significantly reduced in cycling Raptor-deficient progenitors. These data indicate that the impact of mTORC1 deficiency on cell cycle status varies substantially depending on the cell context. In addition, we evaluated the effect of hyperactivation of mTORC1 by Tsc1 deletion on the behavior of T-ALL. Tsc1 deficiency shortened survival, and promoted the cell proliferation, as well as the dissemination of active Notch-driven T-ALL cells in non-hematopoietic tissues. However, strikingly, Raptor deficiency resulted in efficient leukemia eradication. Thus, understanding the cell-context-dependent role of mTORC1 illustrates the potential importance of mTOR signals as therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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Role of cell cycle regulator p19ARF in regulating T cell responses

Cellular Immunology ◽

10.1016/s0008-8749(02)00597-x ◽

2002 ◽

Vol 219 (2) ◽

pp. 119-130 ◽

Cited By ~ 2

Author(s):

Xiaoyan Gao ◽

John Svaren ◽

M. Suresh

Keyword(s):

Cell Cycle ◽

T Cell ◽

T Cell Responses ◽

Cell Responses ◽

Cell Cycle Regulator

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Vpr R36w and R77Q Mutations Alter HIV-1 Replication and Cytotoxicity in T Lymphocytes

10.21203/rs.3.rs-107662/v1 ◽

2020 ◽

Author(s):

Antonio Solis-Leal ◽

Dalton C. Karlinsey ◽

J. Brandon Lopez ◽

Vicente Planelles ◽

Brian D. Poole ◽

...

Keyword(s):

Cell Cycle ◽

T Cell ◽

Cell Cycle Arrest ◽

Wild Type ◽

Helper T Cell ◽

Cycle Arrest ◽

Aids Progression ◽

G2 Cell Cycle Arrest ◽

Hiv 1

Abstract Background: Acquired immunodeficiency syndrome (AIDS) is caused when HIV depletes CD4+ helper T cell levels in infected patients. Distinct AIDS development rates have shown that there are Rapid Progressor (RP) and Long-Term Non-Progressor (LTNP) patients, but the circumstances governing these differences in the kinetics of helper T cell depletion are poorly understood. Mutations in the Viral Protein R (Vpr) gene have been suggested to have a direct impact on helper T cell depletion. Interactions of Vpr with both host and viral factors affect cellular activities such as cell cycle progression and apoptosis. The Vpr mutants R36W and R77Q have been associated with RP and LTNP phenotypes, respectively; however, these findings are still controversial. This study examines the effects that Vpr mutations have in the context of HIV-1 infection of the HUT78 T cell line, using replication-competent CXCR4-tropic virus strains. Results: Our results show a replication enhancement of the R36W mutant accompanied by increased cytotoxicity. Interestingly, the R77Q mutant showed a unique enhancement of apoptosis (measured by Annexin V and TUNEL staining) and G2 cell cycle arrest; these effects were not seen with WT, R36W or Vpr null viruses. Thus, point mutations in Vpr can exhibit profound differences in mechanisms and rates of cell killing. Conclusions: The vpr gene is thought to be an important virulence factor in Human Immunodeficiency Virus type 1 (HIV-1). Vpr polymorphisms have been associated with different rates of AIDS progression. However, there is controversy about the cytopathic and virulence phenotypes of Vpr mutants, with contradictory conclusions about the same mutants. Here, we examine the replication capacity, apoptotic induction, and G2 cell cycle arrest phenotypes of three vpr mutants compared to wild-type HIV-1. One mutant associated with rapid AIDS progression replicated more efficiently and killed cells more rapidly than wild-type HIV-1. Another mutant associated with slow AIDS progression triggered apoptosis more efficiently than wild-type HIV-1 and showed significant levels of G2 cell cycle arrest. These results shed additional light on the role of vpr polymorphisms in T cell killing by HIV-1 and may help to explain the role of Vpr in different rates of AIDS progression.

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Retinoic Acid Receptor Alpha Expression Drives Cell-Cycle Progression and Is Associated with Increased Sensitivity to Retinoids in Peripheral T-Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.1749.1749 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1749-1749

Author(s):

Rebecca L Boddicker ◽

Xueju Wang ◽

Surendra Dasari ◽

Grzegorz S. Nowakowski ◽

Konstantinos N Lazaridis ◽

...

Keyword(s):

Cell Cycle ◽

Retinoic Acid ◽

T Cell ◽

Cell Growth ◽

Cell Lines ◽

Research Funding ◽

Retinoic Acid Receptor ◽

Wild Type ◽

T Cell Lymphomas

Abstract Background: Peripheral T-cell lymphomas (PTCLs) are aggressive non-Hodgkin lymphomas with marked clinical, pathological, and molecular heterogeneity. Outcomes following standard therapy generally are poor; however, few candidate therapeutic targets have been identified for precision medicine approaches. Retinoic acid receptor alpha (RARA) is a transcription factor that modulates cell growth and differentiation in response to natural or synthetic retinoids. Retinoids have been used successfully to treat acute promyelocytic leukemia and some cutaneous T-cell lymphomas (CTCLs). However, the function of RARA and the action of retinoids in PTCL have not been defined. Methods:Based on identification of a PTCL patient with a non-synonymous point mutation, RARA R394Q, identified in the Mayo Clinic Center for Individualized Medicine, we sought to characterize the role of RARA in PTCL cells. To investigate the role of wild-type and mutant RARA, we constructed expression vectors containing either wild-type RARA or RARA R394Q coding sequences, and also used siRNAs targeting RARA to study the role of native RARA expression. Cell lines derived from post-thymic T-cell malignancies were used for in vitro studies, including HuT78 and Mac-1 (both derived from circulating tumor cells from CTCL patients) and Karpas 299 (from an ALK-positive anaplastic large cell lymphoma). Following RARA overexpression or knockdown, we measured cell growth, cell cycle regulation, and sensitivity to synthetic retinoids. In addition, RNA sequencing and pathway analysis were performed to profile the transcriptomic response to retinoids in malignant T cells. Results:In two RARAlow cell lines, Karpas 299 and HuT78, overexpression of wild-type RARA or RARA R394Q significantly increased cell growth (p<0.001), with a greater increase observed from mutant versus wild-type RARA in Karpas 299 (136% of control versus 122%; p=0.04). Accordingly, knockdown of wild-type RARA in the RARAhigh cell line, Mac-1, resulted in a 22% inhibition of cell growth (p=0.0002). This inhibition specifically was associated with G1 cell cycle arrest (120% of control; p=0.004) and decreased protein expression of the G1-S-associated cyclin-dependent kinases, CDK2, CDK4, and CDK6. These kinases were up-regulated by overexpression of RARA in RARAlow HuT78 cells. The relatively RARA-specific retinoid, AM80 (tamibarotene), and the less specific retinoid, all-trans retinoic acid (ATRA), resulted in RARA protein degradation, cell growth inhibition that was both dose-dependent and proportional to baseline RARA expression, G1 arrest, and CDK protein up-regulation. Gene-set enrichment analysis (GSEA) of transcriptome data confirmed that genes down-regulated by AM80 were highly enriched for regulators of cell cycle and particularly G1-S transition. Finally, overexpressing RARA in RARAlow Karpas 299 and HuT78 cell lines significantly increased the ability of AM80 to inhibit CDK2/4/6 expression and cell growth (16% to 23% greater growth inhibition than control; p<0.05). Conclusions:RARA drives cyclin-dependent kinase expression and G1-S transition in malignant T cells, and promotes cell growth. These functions may be enhanced by specific RARA gene mutations. Synthetic retinoids inhibit these functions in a dose-dependent fashion, and are most effective in cells with high RARA expression. These data suggest RARA as a candidate therapeutic target in some PTCL patients. Disclosures Nowakowski: Celgene: Research Funding; Morphosys: Research Funding; Bayer: Consultancy, Research Funding.

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Frequency of B lymphocytes responsive to anti-immunoglobulin.

Journal of Experimental Medicine ◽

10.1084/jem.155.5.1523 ◽

1982 ◽

Vol 155 (5) ◽

pp. 1523-1536 ◽

Cited By ~ 202

Author(s):

A L Defranco ◽

E S Raveche ◽

R Asofsky ◽

W E Paul

Keyword(s):

Cell Cycle ◽

T Cell ◽

B Cells ◽

B Lymphocytes ◽

Resting State ◽

S Phase ◽

Almost All ◽

Antibody Secreting Cells

The frequency of murine B lymphocytes that respond to antibodies directed against membrane IgM was measured. These anti-mu antibodies induced all, or almost all, resting B cells to enlarge over the first 24 h of stimulation. This probably represents the transition from the resting state (G0) to active transit through the cell cycle. In contrast, only a fraction of these cells, approximately 60% for BDF1 mice, continued through the cell cycle into S phase. This is consistent with previous experiments that had suggested there were some types of B cells that did not proliferate in response to anti-mu. The results presented here demonstrate that many, perhaps all, of these nonresponding B cells, both from normal mice and from mice with the xid defect, actually do respond to the presence of anti-mu by going through early parts of the cell cycle. These cells appear to become blocked at some point before the beginning of S phase, perhaps requiring a signal from a T cell or a macrophage to continue through the cell cycle. Thus, the role of antigen may be to prepare all B cells for proliferation. Different subpopulations of B cells may then require different regulatory signals before actually proliferating or before differentiating into antibody-secreting cells.

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