scholarly journals HIF1α–dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells

2011 ◽  
Vol 208 (7) ◽  
pp. 1367-1376 ◽  
Author(s):  
Lewis Z. Shi ◽  
Ruoning Wang ◽  
Gonghua Huang ◽  
Peter Vogel ◽  
Geoffrey Neale ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Fate ◽  
Treg Cell ◽  
Cell Lineage ◽  
Cellular Metabolism ◽  
Treg Cells ◽  
Glycolytic Pathway ◽  
Metabolic Switch ◽  
Glycolytic Activity

Upon antigen stimulation, the bioenergetic demands of T cells increase dramatically over the resting state. Although a role for the metabolic switch to glycolysis has been suggested to support increased anabolic activities and facilitate T cell growth and proliferation, whether cellular metabolism controls T cell lineage choices remains poorly understood. We report that the glycolytic pathway is actively regulated during the differentiation of inflammatory TH17 and Foxp3-expressing regulatory T cells (Treg cells) and controls cell fate determination. TH17 but not Treg cell–inducing conditions resulted in strong up-regulation of the glycolytic activity and induction of glycolytic enzymes. Blocking glycolysis inhibited TH17 development while promoting Treg cell generation. Moreover, the transcription factor hypoxia-inducible factor 1α (HIF1α) was selectively expressed in TH17 cells and its induction required signaling through mTOR, a central regulator of cellular metabolism. HIF1α–dependent transcriptional program was important for mediating glycolytic activity, thereby contributing to the lineage choices between TH17 and Treg cells. Lack of HIF1α resulted in diminished TH17 development but enhanced Treg cell differentiation and protected mice from autoimmune neuroinflammation. Our studies demonstrate that HIF1α–dependent glycolytic pathway orchestrates a metabolic checkpoint for the differentiation of TH17 and Treg cells.

Most human thymic and peripheral-blood CD4+CD25+ regulatory T cells express 2 T-cell receptors

Blood ◽  
2006 ◽  
Vol 108 (13) ◽  
pp. 4063-4070 ◽  
Author(s):  
Heli Tuovinen ◽  
Jukka T. Salminen ◽  
T. Petteri Arstila
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treg Cell ◽  
Flow Cytometric Analysis ◽  
Cell Lineage ◽  
Cell Receptor ◽  
Treg Cells ◽  
Allelic Exclusion ◽  
Dual Specificity ◽  
Functional Consequences

Abstract Lack of allelic exclusion in the T-cell receptor (TCR) α locus gives rise to 2 different TCRs in 10% to 30% of all mature T cells, but the significance of such dual specificity remains controversial. Here we show that human CD4+CD25+ regulatory T (Treg) cells express 2 distinct Vα chains and thus 2 TCRs at least 3 times as often as other T cells. Extrapolating from flow cytometric analysis using Vα2-, Vα12-, and Vα24-specific monoclonal antibodies (mAbs), we estimated that between 50% and 99% of the CD25+ Treg cells were dual specific, as compared with about 20% of their CD25– counterparts. Moreover, both TCRs were equally capable of transmitting signals upon ligation. Cells with 2 TCRs also expressed more FOXP3, the Treg-cell lineage specification factor, than cells with a single TCR. Our findings suggest that expression of 2 TCRs favors differentiation to the Treg-cell lineage in humans and raise the question of the potential functional consequences of dual specificity.

scholarly journals Dll4–Notch signaling in Flt3-independent dendritic cell development and autoimmunity in mice

2012 ◽  
Vol 209 (5) ◽  
pp. 1011-1028 ◽  
Author(s):  
Fabienne Billiard ◽  
Camille Lobry ◽  
Guillaume Darrasse-Jèze ◽  
Janelle Waite ◽  
Xia Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Dendritic Cell ◽  
Notch Signaling ◽  
Cell Fate ◽  
Treg Cell ◽  
De Novo ◽  
Alternative Pathway ◽  
Treg Cells ◽  
Cell Development

Delta-like ligand 4 (Dll4)–Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4–Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4+CD25+FoxP3+ regulatory T cells (Treg cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural Treg cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4−CD8−c-kit+CD44+CD25− (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4+ T cells into Treg cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of Treg cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a Treg cell–mediated mechanism and inhibits CD8+ T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased Treg cell numbers in the pancreas-draining lymph nodes. These results identify Dll4–Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated Treg cell homeostasis and autoimmunity.

scholarly journals Graded RhoA GTPase Expression in Treg Cells Distinguishes Tumor Immunity From Autoimmunity

2021 ◽  
Vol 12 ◽  
Author(s):  
Khalid W. Kalim ◽  
Jun-Qi Yang ◽  
Vishnu Modur ◽  
Phuong Nguyen ◽  
Yuan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treg Cell ◽  
Tumor Immunity ◽  
Th17 Cell ◽  
Treg Cells ◽  
Effector T Cells ◽  
Conditional Knockout ◽  
Cell Plasticity ◽  
Cell Homeostasis

RhoA of the Rho GTPase family is prenylated at its C-terminus. Prenylation of RhoA has been shown to control T helper 17 (Th17) cell-mediated colitis. By characterizing T cell-specific RhoA conditional knockout mice, we have recently shown that RhoA is required for Th2 and Th17 cell differentiation and Th2/Th17 cell-mediated allergic airway inflammation. It remains unclear whether RhoA plays a cell-intrinsic role in regulatory T (Treg) cells that suppress effector T cells such as Th2/Th17 cells to maintain immune tolerance and to promote tumor immune evasion. Here we have generated Treg cell-specific RhoA-deficient mice. We found that homozygous RhoA deletion in Treg cells led to early, fatal systemic inflammatory disorders. The autoimmune responses came from an increase in activated CD4+ and CD8+ T cells and in effector T cells including Th17, Th1 and Th2 cells. The immune activation was due to impaired Treg cell homeostasis and increased Treg cell plasticity. Interestingly, heterozygous RhoA deletion in Treg cells did not affect Treg cell homeostasis nor cause systemic autoimmunity but induced Treg cell plasticity and an increase in effector T cells. Importantly, heterozygous RhoA deletion significantly inhibited tumor growth, which was associated with tumor-infiltrating Treg cell plasticity and increased tumor-infiltrating effector T cells. Collectively, our findings suggest that graded RhoA expression in Treg cells distinguishes tumor immunity from autoimmunity and that rational targeting of RhoA in Treg cells may trigger anti-tumor T cell immunity without causing autoimmune responses.

Regulation of Hematopoietic Colony Forming Cells by CD4+CD25+ T Cells: A Role for T Regulatory Cells in Hematopoiesis?.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3181-3181
Author(s):  
Maite Urbieta ◽  
Isabel Barao ◽  
Monica Jones ◽  
William J. Murphy ◽  
Robert B. Levy
Keyword(s):  
T Cells ◽  
Growth Factors ◽  
T Cell ◽  
Treg Cell ◽  
Mhc Class Ii ◽  
Progenitor Cell ◽  
Cell Activity ◽  
Class Ii ◽  
Treg Cells

Abstract CD4+CD25+ T cells (Treg) comprise a small population within the normal peripheral CD4 T cell compartment. Their primary physiological role appears to be the regulation of autoimmune responses, however, in recent years it has been established that they can modulate anti-tumor as well as transplantation responses. Treg cells have been found to exert their affects on multiple types of immunologically relevant cells including CD4, CD8 and NK populations. Although model dependent, cytokines including TGFβ and IL-10 have been identified as mediators of this population’s regulatory activity and ex-vivo, the inhibition effected is generally contact dependent. Based upon the expanding application of Treg cells in stem cell transplants for the control of GVHD, rejection (HVG) and GVL responses, we hypothesized that following T cell receptor engagement and activation in recipients, CD4+CD25+ cells may modulate hematopoietic responses via production of effector cytokines. To address this question, various populations of CD4+CD25+ T cells were initially co-cultured with unfractionated syngeneic bone marrow cells (BMC) for 24–48 hours in medium supplemented with growth factors to maintain progenitor cell (i.e. CFU) function. Following co-culture, cells were collected and replated in triplicate in methylcellulose containing medium together with hematopoietic growth factors and five-seven days later, colonies were counted. CD4+CD25+ T cells were purified from BALB/c or B6–CD8−/− mice which were then activated for 3–8 days with anti-CD3/CD28 beads (a gift of Dr. B. Blazar, U. Minn.) These cells inhibited syngeneic CFU-IL3 colony ($25 cells) formation at ratios as low as 2:1 and 0.5:1 CD4+CD25+: BMC. Notably, Tregs from B6-CD8−/− mice exhibited comparable inhibition of allogeneic (BALB/c) CFU-IL3. Non-activated CD4+CD25+ T cells co-cultured with BMC did not exhibit this inhibitory activity nor did CD4+CD25− cells which contaminated (<10%) CD4+CD25+ populations. Activated Treg cells were also found to inhibit the production of CFU-HPP, a multi-potential marrow progenitor cell population. Contact dependency was found to be required for this effect as separation of activated CD4+CD25+ T cells from BMC “targets” in trans-well cultures abrogated inhibition. Prior depletion of CD25+ cells in vivo resulted in increases in CFU-GM 7–9 days after syngeneic BMT in mice suggesting that Tregs can inhibit hematopoietic reconstitution in vivo. To examine a potential contribution of TGFβ in this model, neutralizing anti-TGFβ mab was added during CD4+CD25+ T cell + BMC co-culture. The inhibition of CFU activity was abrogated in the presence of this antibody. To begin investigating the role of MHC class II molecules in this Treg cell activity, c-kit+ enriched (>85%) BMC from B6-MHC class II KO and B6-wt mice were co-cultured with B6 Treg cells from CD8−/− mice. In contrast to B6-wt c-kit enriched populations, CFU inhibition was not detected against the MHC class II deficient c-kit enriched BMC population. Antibody experiments are in progress to determine if cognate interaction is required between c-kit enriched cells and CD4+CD25+ T cells. In summary, this is the first report demonstrating that CD4+CD25+ T cells can alter hematopoietic progenitor cell activity. We hypothesize that membrane bound TGFβ may participate in effecting such regulation via direct Treg cell interactions with progenitor cell populations.

Inhibiting IL-2 Signaling and the Regulatory T-Cell Pathway Using Computationally Designed Novel Peptides

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3875-3875
Author(s):  
Tammy Price-Troska ◽  
David Diller ◽  
Alexander Bayden ◽  
Mark Jarosinski ◽  
Joseph Audies ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Function ◽  
Treg Cell ◽  
Immune Cell ◽  
Conflicts Of Interest ◽  
Interleukin 2 ◽  
Computational Design ◽  
Treg Cells ◽  
Cell Numbers

Abstract Regulatory T-cells (TREG) are the gateway to immune function and typically regulate immune cell activation. Cytokines, including interleukin-2 (IL-2), induce T-cell differentiation and promote a regulatory phenotype. Once activated via the IL-2 receptor (IL-2R), a cascade of events in T-cells initiate signal transducer and activator of transcription 5 (STAT5) and Forkhead box P3 (FOXP3) activation which appear to function as important regulators of this immunologic pathway and promote the development and function of TREG cells. In non-Hodgkin lymphoma (NHL), we have found that intratumoral TREG cells are increased in number and suppress immune function. In previous work, we have found that TREG cells inhibit T-cell proliferation, suppress cytokine production and limit effector cell cytotoxicity. We have also shown that increased serum levels of soluble sIL-2Rα is a prognostic factor in NHL and that sIL-2Rα can bind to IL-2 and promote its signaling thereby increasing TREG cell numbers. In this study, we developed a strategy to inhibit the binding of IL-2 to sIL-2Rα with the goal of suppressing the induction of FOXP3 and decreasing TREG cell numbers. To do this, we developed peptides designed to disrupt the interaction between IL2 and sILRα. In collaboration with CMDBioscienceSM, we developed and analyzed 22 peptide compounds derived by structure-based computational design. Initially, we screened each peptide at increasing concentrations using an ELISA assay to test the inhibition of IL-2/IL-2Rα binding by the solubilized peptide. Candidate peptides were then further tested using upregulation of pSTAT5 and FOXP3 in T-cells measured by flow cytometry as a measure of inhibition of IL-2 signaling. The peptides were developed according to different design hypotheses and grouped into different families; the screening ELISA results indicated 4 promising peptides that inhibited IL2/IL2Rα binding (up to 100% inhibition; max peptide concentration of 100uM). These peptides were then used to determine their effect on STAT5 and FOXP3 expression. A lead candidate peptide consistently reduced the expression of FOXP3 and STAT5 expression compared to cells not exposed to peptide. Use of the peptide to disrupt IL-2 signaling inhibited the development of cells with a TREG phenotype. We conclude that structure-based peptide design can be used to identify novel peptide inhibitors that block IL-2/IL-2Rα signaling and inhibit STAT5 and FOXP3 upregulation. These peptides could be used as new therapeutic agents to limit immune suppression by TREG cells and promote a more effective anti-tumor immune response in NHL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting of Cdc42 GTPase in regulatory T cells unleashes anti-tumor T cell immunity

2021 ◽  
Author(s):  
Khalid W Kalim ◽  
Jun-Qi Yang ◽  
Mark Wunderlich ◽  
Vishnu Modur ◽  
Phuong Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treg Cell ◽  
Cell Function ◽  
Treg Cells ◽  
T Cell Response ◽  
T Cell Immunity ◽  
Cell Plasticity ◽  
Effector T Cell ◽  
Cell Immunity

Regulatory T (Treg) cells play an important role in maintaining immune tolerance through inhibiting effector T cell function. In the tumor microenvironment, Treg cells are utilized by tumor cells to counteract effector T cell-mediated tumor killing. Targeting Treg cells may thus unleash the anti-tumor activity of effector T cells. While systemic depletion of Treg cells can cause excessive effector T cell responses and subsequent autoimmune diseases, controlled targeting of Treg cells may benefit cancer patients. Here we show that Treg cell-specific heterozygous deletion or pharmacological targeting of Cdc42 GTPase does not affect Treg cell numbers but induces Treg cell plasticity, leading to anti-tumor T cell immunity without detectable autoimmune reactions. Cdc42 targeting potentiates an immune checkpoint blocker anti-PD-1 antibody-mediated T cell response against mouse and human tumors. Mechanistically, Cdc42 targeting induces Treg cell plasticity and unleashes anti-tumor T cell immunity through carbonic anhydrase I-mediated pH changes. Thus, rational targeting of Cdc42 in Treg cells holds therapeutic promises in cancer immunotherapy.

Regulatory T Cells Enhance the Allogeneic Activity of Endothelial-Specific Cytotoxic T Lymphocytes – Protective Effect of Defibrotide

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4693-4693
Author(s):  
Guenther Eissner ◽  
Isabel Hartmann ◽  
Altug Kesikli ◽  
Silvia Haffner ◽  
Tanja Sax ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
T Cell ◽  
T Lymphocytes ◽  
Treg Cell ◽  
Interleukin 2 ◽  
Treg Cells ◽  
Protective Function ◽  
Phenotypic Quality

Abstract Abstract 4693 BACKGROUND: Damage to the vascular endothelium is the primary event of transplant related complications and often precedes loss of organ function. Depending on the amount of co-stimulatory signals, endothelial cells can either act as stimulating or inhibiting antigen presenting cells (APC). On the other hand, numerous data indicate that CD4+CD25+FoxP3+ T cells (Treg cells) can attenuate alloresponses of conventional T lymphocytes against classical APC and thus qualify for clinical use in various transplant settings. However, it is unknown whether Treg cells also influence T cell – endothelial cell interactions. Defibrotide (DF) is a polydisperse mixture of single-stranded deoxyribonucleotides with anti-thrombotic and anti-inflammatory activity, known to modulate the antigenicity of vascular endothelial cells. METHODS: CD8+ T lymphocytes (CTL) were isolated by magnetic microbead separation of peripheral blood mononuclear cells (PBMC) from healthy human blood donors and stimulated with mito-inactivated cells of a human microvascular endothelial cell line (CDC/EU.HMEC-1, further referred to as EC) and other primary and transformed micro- and macrovascular ECs for 7 days in the presence of interleukin 2 (IL-2). Treg cells from the CTL donor were prepared by CD4 (untouched) and a double CD25 microbead separation as well as a CD127bright depletion, followed by anti-CD3/CD28 expansion in the presence of IL-2 and a phenotypic quality control. Treg cells were added to the CTL-EC co-culture (1:1:1) prior to 51Cr release or flow cytometric cytotoxicity assays. Additionally, Treg cells were also tested for their capacity to influence CTL lysis of Epstein-Barr-Virus-transformed B-LCL, which as classical APC were HLA-matched to the HMEC. Furthermore, EC targets were incubated in the presence or absence of DF (25μM) for 24 hrs to assess the drug's protective function on the allogenicity of EC. RESULTS: EC-stimulated CTL showed a specific MHC class I-restricted target lysis. Addition of Treg cells prior to the cytotoxicity assay and during the afferent immune phase surprisingly increased EC lysis by CTL. In contrast, Treg cells alone did not show any lytic activity against EC. As a control, conventional CD4+CD25- T cells did not influence CTL activity either. Treg cell-mediated enhancement was endothelial-specific, since B-LCL lysis was not influenced. Further subpopulation analysis revealed the existence of CD8+/CD28-/CD57+ CTL, requiring cell-to-cell contact with Treg cells for their increased activity towards EC. Importantly, DF could almost fully protect EC against lysis by allogeneic CD28- CTL and the Treg cell-mediated enhancement. Of note, DF exclusively protected EC and did influence T cell function nor viability, suggesting a strong tropism for the endothelial cell type. CONCLUSION: There is no doubt about the potential therapeutic efficacy of Treg cells to ameliorate outcome of allogeneic transplants, but the endothelium might require additional protective interventions to prevent specific alloreactivity, such as DF. Disclosures: Eissner: Gentium, Sp.A.: Consultancy. Iacobelli:Gentium SpA: Employment.

TOR Complex 1 and TOR Complex 2 Differentially Regulate Effector and Regulatory T Cell Differentiation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 675-675 ◽  
Author(s):  
Greg M Delgoffe ◽  
Thomas P. Kole ◽  
Yan Zheng ◽  
Bo Xiao ◽  
Paul F. Worley ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Fate ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Lineage ◽  
Lineage Commitment ◽  
Th2 Cells ◽  
Double Positive

Abstract Effector T cell lineage commitment is determined by the integration of multiple and sometimes opposing signals. Our lab has identified mTOR, an evolutionarily conserved serine/threonine kinase, as a crucial protein dictating the outcome of T cell fate in response to antigen. To do this we utilized a Cre-lox system to conditionally delete mTOR in T cells. In such mice, although mTOR is deleted in the double-positive stage, lymphocyte populations in the spleen and the periphery are comparable to wild-type mice. T cells lacking mTOR proliferate more slowly but secrete appropriate levels of IL-2 upon initial stimulation. However, such cells fail to differentiate into Th1, Th2 or Th17 effector T cells under the appropriate skewing conditions. This failure to differentiate is the result of decreases in appropriate STAT activation and the concomitant lack of upregulation in lineage specific transcription factors. Notably, under normally activating conditions, T cells lacking mTOR preferentially differentiate into Foxp3+ regulatory cells. Supporting this observation, mTOR deficient T cells display hyperactive Smad3 activation, even in the absence of exogenous TGF-β. mTOR signals through two known signaling complexes, TORC1 and TORC2. TORC1 contains Rheb, mTOR, GβL, and raptor, while TORC2 contains mSin1, mTOR, GβL, and rictor. In order to determine the specific role of TORC1 in T cell lineage commitment we conditionally deleted Rheb in T cells. Upon activation such cells fail to phosphorylate the TORC1 substrate S6K-1 while demonstrating normal TORC2 activity. As was the case for the mTOR−/− T cells, Rheb−/− T cells fail to differentiate into Th1 and Th17 cells when skewed in vitro. However, unlike mTOR−/− T cells, the Rheb deficient T cells are capable of becoming Th2 cells. In spite of lacking TORC1 activity, T cells lacking Rheb do not spontaneously develop into Foxp3+ cells. Such observations implicate a specific and novel role for Rheb in regulating T cell lineage commitment. Overall, our data identify mTOR as a regulator of T cell lineage commitment through which TORC1 and TORC2 signaling differentially regulate T cell fate. These findings support a novel paradigm whereby T cell activation induces a default pathway of differentiation to regulatory T cells and that TORC2 signaling is required to divert differentiation to appropriately programmed effector lineages.

Role of regulatory T-cells in autoimmunity

2009 ◽  
Vol 116 (8) ◽  
pp. 639-649 ◽  
Author(s):  
Richard J. Mellanby ◽  
David C. Thomas ◽  
Jonathan Lamb
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Treg Cell ◽  
Cell Function ◽  
Cell Subset ◽  
Treg Cells ◽  
Self Tolerance

There has been considerable historical interest in the concept of a specialist T-cell subset which suppresses over-zealous or inappropriate T-cell responses. However, it was not until the discovery that CD4+CD25+ T-cells had suppressive capabilities both in vitro and in vivo that this concept regained credibility and developed into one of the most active research areas in immunology today. The notion that in healthy individuals there is a subset of Treg-cells (regulatory T-cells) involved in ‘policing’ the immune system has led to the intensive exploration of the role of this subset in disease resulting in a number of studies concluding that a quantitative or qualitative decline in Treg-cells is an important part of the breakdown in self-tolerance leading to the development of autoimmune diseases. Although Treg-cells have subsequently been widely postulated to represent a potential immunotherapy option for patients with autoimmune disease, several studies of autoimmune disorders have demonstrated high numbers of Treg-cells in inflamed tissue. The present review highlights the need to consider a range of other factors which may be impairing Treg-cell function when considering the mechanisms involved in the breakdown of self-tolerance rather than focussing on intrinsic Treg-cell factors.

831 E3 ubiquitin ligase Cbl-b deficient CD8+ T cells overcome Treg cell-mediated suppression through IFN-γ and induce robust anti-tumor immunity

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A883-A883
Author(s):  
SeongJun Han ◽  
Zhe Qi Liu ◽  
Douglas Chung ◽  
Michael St Paul ◽  
Carlos Garcia-Batres ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Treg Cell ◽  
Ubiquitin Ligase ◽  
Cd8 T Cells ◽  
Treg Cells ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
Ifn Γ

BackgroundAdoptive T cell therapy (ACT) is reaching its potential in multiple malignancies. However, anti-tumor T cell responses can be attenuated by suppressive cells in the tumor microenvironment, such as CD4+FoxP3+ regulatory T (Treg) cells. Depletion of Treg cells can be technically challenging in ACT and may be associated with unwanted adverse effects. Alternatively, studies suggest that specific modifications in T cell signaling network may render T cells resistant to regulation by Treg cells. Here, we investigated the role of Casitas B- Lineage Lymphoma-b (Cbl-b), an E3 ubiquitin ligase and a negative regulator of TCR signaling pathways, in rendering CD8+ T cells resistant to the effects of Treg cells to bolster ACT.MethodsIn vitro stimulated Cbl-b+/+ or Cbl-b-/- Thy1.1+ P14 TCR-transgenic CD8+ T cells were adoptively transferred into B16-gp33 melanoma-bearing Thy1.2+ FoxP3-GFP/DTR transgenic mice treated with or without diphtheria toxin (n = 15). Tumor size and overall survival were measured. Congenically labelled T cells from tumor, draining lymph node, and spleen were comprehensively profiled using flow cytometry. To further examine the biological mechanism of Treg resistance, we performed in vitro Treg suppression assays and RNA-sequencing.ResultsAdoptively transferred tumor-specific Cbl-b-/- effector CD8+ T cells mediated superior control over tumor growth and increased overall survival in comparison to the wild-type counterpart. Depletion of FoxP3+ cells increased the quantity and percentage of CD25+ 4-1BB+ expressing P14 Thy1.1+ CD8+ T cells in the tumor, whereas the effect of FoxP3+ cell depletion was negligible with Cbl-b deficient CD8+ T cells. Cbl-b deficiency also attenuated sensitivity to Treg cell-mediated suppression in vitro. Transcriptomic analyses suggested that Cbl-b regulates pathways associated with cytokine production and cellular proliferation. Specifically, hyper-secretion of IFN-γ by Cbl-b deficient CD8+ T cells attenuated suppression by Treg cells. In murine models of adoptive T cell therapy, Cbl-b deficient CD8+ T cells were less susceptible to suppression by Treg cells in the tumor through the effects of IFN-γ.ConclusionsWe demonstrate that adoptively transferred effector CD8+ T cells are susceptible to regulation by Treg cells in the tumor, and that ablation of Cbl-b abrogates Treg cell-mediated suppression. We highlight the therapeutic implications of targeting Cbl-b in the context of ACT.AcknowledgementsWe would like to thank Dr. Tak Mak and Dr. Naoto Hirano for their suggestions and insights for this project.

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