IL-9 Production by Regulatory T Cells Recruits Mast Cells That Are Essential for Regulatory T Cell-Induced Immune-Suppression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2782-2782
Author(s):  
Anna Maria Wolf ◽  
Dominik Wolf ◽  
Andrew McKenzie ◽  
Marcus Maurer ◽  
Alexander R Rosenkranz ◽  
...  
Keyword(s):  
T Cells ◽  
Mast Cells ◽  
Regulatory T Cells ◽  
Adoptive Transfer ◽  
Conflicts Of Interest ◽  
Cell Populations ◽  
Perfect Agreement ◽  
Immunosuppressive Cell

Abstract Abstract 2782 Tipping the balance between effector and regulatory cell populations is of critical importance in the pathogenesis of various autoimmune disorders. Both, mast cells (MC) and regulatory T cells (Treg) have gained attention as immunosuppressive cell populations. To investigate a possible interaction, we used the Th1- and Th17-dependent model of nephrotoxic serum nephritis (NTS), in which both MC and Treg have been shown to play a protective role. We recently provided evidence that adoptive transfer of wild-type (wt) Treg into wt recipients almost completely prevents development of NTS. We here show that Treg transfer induces a profound increase of MC in the kidney draining lymph nodes (LN). In contrast, transfer of wt Treg into animals deficient in MC, which are characterized by an exaggerated susceptibility to NTS, do not prevent acute renal inflammation. Blocking the pleiotropic cytokine IL-9, which is known to be critically involved in MC recruitment and proliferation, by means of an antagonizing monoclonal antibody in animals receiving wt Treg abrogated protection from NTS. Moreover, we provide clear evidence that Treg-derived IL-9 is critical for MC recruitment as mediators of their full immune-suppressive potential, as adoptive transfer of IL-9 deficient Treg failed to protect from NTS. In line with our hypothesis, absence of Treg-derived IL-9 does not induce MC accumulation into kidney-draining LN, despite the fact that IL-9 deficiency does not alter the general suppressive activity of Treg, as shown by in vitro testing of their functional capacities. Finally, we observed a significantly decreased expression of the MC chemoattractant Cxcl-1 in the LN of mice receiving IL-9 deficient Treg as compared to mice receiving wt Treg or control CD4+CD25− T cells, which might at least in part explain the deficient MC recruitment under these conditions. In summary, our data provide the first evidence that the immunosuppressive effects of adoptively transferred Treg depend on IL-9-mediated recruitment of MC to the kidney draining LN in NTS. This data is in perfect agreement with our previous report showing that CCR7-mediated LN occupancy of Treg is a prerequisite for their immune-suppressive potential and furthers adds a piece of information to the functional understanding of the in vivo anti-inflammatory effects of Treg. Disclosures: No relevant conflicts of interest to declare.

Gvhd Severity Is Regulated by the Adoptive Transfer Low Numbers of NKT Cells by a Mechanism Distinct From CD4+CD25+ Regulatory T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 229-229
Author(s):  
Dennis Leveson-Gower ◽  
Janelle Olson ◽  
Emanuela I Sega ◽  
Jeanette Baker ◽  
Robert Zeiser ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Adoptive Transfer ◽  
Conflicts Of Interest ◽  
Serum Levels ◽  
Nkt Cells ◽  
Donor Type ◽  
Ifn Γ ◽  
The Impact

Abstract Abstract 229 NKT cells, a subset of which are CD1d reactive, play an important immunoregulatory role in suppressing dysfunctional immune reactions, including graft-versus-host disease (GVHD). To explore the biological activity and mechanism of donor-type NKT in suppression of GVHD, we utilized highly purified (>95%) populations of donor (C57Bl6; H-2b) NKT (DX5+TCR+CD4+) cells adoptively transferred into lethally irradiated recipient (Balb/c; H-2d) animals with T cell depleted bone marrow (TCD-BM). Highly purified (>95%) NKT cells (5.5×105) from luciferase positive (luc+) C57BL/6 mice were infused into lethally irradiated Balb/c recipients with TCD-BM(5×106) from wild-type (WT) C57BL/6 mice, and the animals were monitored by bioluminescence imaging (BLI). By day 4 after transfer, an NKT derived signal was observed in spleen and lymph node (LN) sites, and between days 7 and 10, NKT had also migrated to the skin. Total photons emitted peaked near day 25 after transplantation, followed by a steady decline. To assess the impact of donor-type NKT cells on GVHD induction by conventional CD4+ and CD8+ T cells (Tcon), we co-transferred various doses of highly purified WT NKT at day 0 with TCD-BM, followed by 5×105 luc+Tcon/animal on day 2. As few as 2.5×104 NKT cells significantly improved survival of mice receiving 5×105 Tcon. Animal survival with Tcon only was 20% and for Tcon with NKT cells was 74%(p=0.0023). In contrast to what is observed with CD4+CD25+FoxP3+ regulatory T cells (Treg), the NKT cells did not suppress Tcon proliferation assayed by both in vivo BLI and in a mixed-leukocyte reaction. Analysis of serum cytokines with or without 2.5×104 NKT, following HCT with TCD-BM and Tcon, indicated the addition of NKT cells resulted in elevated levels of INF-γ, IL-5, and IL-6 in serum; significant differences were not observed in serum levels of IL-2, IL-4, IL-10, IL-17, or TNF-α. Intracellular levels of cytokines in Tcon were analyzed from the same groups. At 8 days after HCT, mice receiving NKT had fewer TNFα-positive cells in LNs (CD4: 45% to 27%; CD8 36% to 24%); by day 11, however, TNFαa levels between groups were equivalent. IFN-γ levels, which were high in both NKT treated and untreated groups at day 8 (85%-95%), decreased significantly in NKT treated mice by day 11 (CD4: 40%; CD8: 43%), but were abundant in Tcon only mice (CD4: 78%; CD8: 80%) (p=.0001). No significant changes were found in the intracellular levels of IL-2, IL-4, IL-5, IL-10, or IL-17 of Tcon in the presence or absence of NKT cells. NKT from both IL-4 -/- and IFN-γ -/- mice were less effective at suppressing GVHD than WT NKT, implicating these cytokines in the suppressive mechanism. Finally, we found that NKT do not have a major impact on the graft-versus-tumor effect of Tcon against a luc+ BCL-1 tumor. These studies indicate that NKT persist in vivo upon adoptive transfer and suppress GVHD, even at extremely low cell numbers, which is important given the relative paucity of this cell population. The mechanisms of GVHD suppression appear to be distinct to those of Treg and involve the production of IL-4 and IFN-γ by NKT resulting in a decrease in Tcon, which produce pro-inflamatory cytokines. Disclosures: No relevant conflicts of interest to declare.

Strong CD28 Costimulation Suppresses Induction of Regulatory T Cells From Naïve Precursors through Lck Signaling.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3728-3728
Author(s):  
Kenrick Semple ◽  
Antony Nguyen ◽  
Yu Yu ◽  
Claudio Anasetti ◽  
Xue-Zhong Yu
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Conflicts Of Interest ◽  
Graft Versus Host ◽  
Cell Immunity ◽  
Cd28 Costimulation ◽  
Insight Into

Abstract Abstract 3728 CD28 costimulation is required for the generation of naturally-derived regulatory T cells (nTregs) in the thymus through Lck-signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naïve CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25+Foxp3+) from naïve CD4 T cells (CD25−Foxp3−) by TCR-stimulation with additional TGFβ in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. By using a series of transgenic mice on CD28-deficient background that bears WT CD28 or mutated CD28 in its cytosolic tail incapable of binding to Lck, PI3K or Itk, we found that CD28-mediated Lck-signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versus-host disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide the rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28-signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CD39+ Regulatory T Cells Attenuate Lipopolysaccharide-Induced Acute Lung Injury via Autophagy and the ERK/FOS Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Cen Chen ◽  
Xinying Li ◽  
Chuling Li ◽  
Jiajia Jin ◽  
Donghui Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lung Injury ◽  
Regulatory T Cells ◽  
Lung Injury ◽  
Adoptive Transfer ◽  
Peripheral Blood ◽  
Novel Method ◽  
Clinical Prevention

Acute respiratory distress syndrome (ARDS) is characterized by an uncontrollable cytokine storm, which is associated with high mortality due to lack of effective treatment. Regulatory T cells (Tregs) play an indispensable role in maintaining immune homeostasis and CD39 is considered as a functional cell marker of Tregs. In this study, we aimed to evaluate the effect of CD39+ Tregs on acute lung injury (ALI) and investigate the frequency of CD39+ Tregs in ARDS patients. We found that after lipopolysaccharide (LPS) treatment, CD39−/− mice exhibited more severe inflammation and wild type (WT) mice exhibited a decreased frequency of CD39+ Tregs in the peripheral blood. Furthermore, CD39+ Tregs had a protective effect on LPS-induced inflammation in vitro and the adoptive transfer of CD39+ Tregs had a therapeutic effect on ALI in vivo. We further sought to explore the mechanisms that affect CD39 expression on Tregs. LPS-induced inflammation in the lung impaired the immunosuppressive effect of Tregs via the autophagy-mediated downregulation of CD39. In addition, CD39 induced the expression of itself in Tregs via activating the ERK1/2-FOS pathway. Consistent with this finding, the frequency of CD39+ Tregs was also decreased in the peripheral blood of ARDS patients and was positively correlated with disease severity. Our results suggested that the adoptive transfer of CD39+ Tregs may provide a novel method for the clinical prevention and treatment of ARDS.

Differential Targeting of Signaling Pathways to Selectively Expand Mouse Regulatory T Cells While Inhibiting Conventional T Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2158-2158
Author(s):  
Atsushi Satake ◽  
Amanda M Schmidt ◽  
Angela Archambault ◽  
Gregory F Wu ◽  
Taku Kambayashi
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Signaling Pathways ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Negative Effects ◽  
Tcr Signaling

Abstract Abstract 2158 Regulatory T cells (Tregs) are suppressive T cells with therapeutic potential for ameliorating T cell-mediated diseases. Thus, there has been great interest in revealing the mechanisms by which Tregs proliferate. Recently, we reported that TCR signaling is partially dispensable for Treg proliferation in vivo when exogenous IL-2 is administered. Based on this data, we hypothesized that when given in conjunction with IL-2, pharmacological inhibition of TCR signaling might allow Tregs to expand while simultaneously inhibiting conventional T cell (Tconv) proliferation. Using mutant mice with defective TCR-mediated PLCγ activation, we found that the activation of PLCγ is dispensable for IL-2-mediated Treg proliferation. In contrast, costimulation-derived mTOR signaling was required for IL-2-induced Treg proliferation. We next used Cyclosporine A (CSA; calcineurin inhibitor) and rapamycin (mTOR inhibitor) to differentially target these signaling pathways. Consistent with our hypothesis, while both CSA and rapamycin suppressed antigen-specific Tconv proliferation, only CSA permitted IL-2-induced Treg expansion in vitro and in vivo. Rapamycin, however, did increase the overall Treg:Tconv ratio due to its negative effects on Tconv survival. Given that CSA inhibited antigen-specific Tconv proliferation while maintaining IL-2-induced Treg expansion, we hypothesized that the combination of CSA and IL-2 would be beneficial for attenuating T cell-mediated disease. Indeed, CSA synergized with IL-2 in protection against experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Surprisingly, however, the administration of CSA blocked whereas rapamycin augmented the beneficial effect of IL-2 in graft-versus-host disease (GVHD). These differences potentially results from the overt TCR stimulation that Tregs would receive in the allogeneic (GVHD) vs. syngeneic (EAE) environment. Moreover, inducible Treg (iTreg) generation from allogeneic MHC-stimulated naïve Tconvs contributes greatly to the Treg pool during GVHD. This was consistent with our data showing that rapamycin promotes iTreg generation and allows TCR-enhanced Treg proliferation, whereas CSA inhibited both of these processes. Thus, depending on the disease setting, the signaling pathways contributing to expansion of the Treg pool need to be carefully considered and specifically targeted to increase the Treg:Tconv ratio in treatment of T cell-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

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 Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 877-885 ◽  
Author(s):  
Y Kanakura ◽  
H Thompson ◽  
T Nakano ◽  
T Yamamura ◽  
H Asai ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tissue Type ◽  
Cell Populations ◽  
Proliferative Potential ◽  
Heparin Binding ◽  
Peritoneal Mast Cells ◽  
Proliferative Ability

Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of [35S] sulfate predominantly into heparin proteoglycans. When PMC purified to greater than 99% purity were cultured in methylcellulose with IL-3 and IL-4, approximately 25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate [35S] proteoglycans. When “MMC-like” cultured mast cells derived from WBB6F1-+/+ PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1- W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these “second generation PMC” formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal +/+ PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells' proliferative ability.

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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