scholarly journals T-cell function is partially maintained in the absence of class IA phosphoinositide 3-kinase signaling

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2894-2902 ◽  
Author(s):  
Jonathan A. Deane ◽  
Michael G. Kharas ◽  
Jean S. Oak ◽  
Linda N. Stiles ◽  
Ji Luo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cell Function ◽  
Pi3k Signaling ◽  
Regulatory Subunits ◽  
Cell Functions ◽  
Phosphoinositide 3 Kinase

Abstract The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85α, p55α, p50α, and p85β. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA–deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.

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 TRPM2 Is Not Required for T-Cell Activation and Differentiation

2022 ◽  
Vol 12 ◽  
Author(s):  
Niels C. Lory ◽  
Mikolaj Nawrocki ◽  
Martina Corazza ◽  
Joanna Schmid ◽  
Valéa Schumacher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Transient Receptor Potential ◽  
Cell Function ◽  
Intestinal Inflammation ◽  
Activation Markers

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2-/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2-/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2-/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2-/- mice and after transfer of WT and Trpm2-/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2-/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

scholarly journals Characterizing T Cells in SCID Patients Presenting with Reactive or Residual T Lymphocytes

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Atar Lev ◽  
Amos J. Simon ◽  
Luba Trakhtenbrot ◽  
Itamar Goldstein ◽  
Meital Nagar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Severe Combined Immunodeficiency ◽  
Cell Activity ◽  
Mhc Ii ◽  
Cell Functions ◽  
Circulating T Cells

Introduction. Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms.Methods. Here we compared T-cell functions including the number of circulating CD3+T cells,in vitroresponses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells.Results. MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs.Conclusion. SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

scholarly journals Genetic control of immune response to myoglobin. Ir gene function in genetic restriction between T and B lymphocytes.

1982 ◽  
Vol 156 (5) ◽  
pp. 1486-1501 ◽  
Author(s):  
Y Kohno ◽  
J A Berzofsky
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
High Responder ◽  
Gene Control ◽  
Gene Defect ◽  
Genetic Restriction ◽  
Low Responder

We studied the genetic restrictions on the interaction between T cells, B cells, and antigen-presenting cells (APC) involved in the H-2-linked Ir gene control of the in vitro secondary antibody response to sperm whale myoglobin (Mb) in mice. The B cells in this study were specific for Mb itself, rather than for a hapten unrelated to the Ir gene control, as in many previous studies. Low responder mice immunized in vivo with Mb bound to an immunogenic carrier, fowl gamma globulin (F gamma G), produced B cells competent to secrete anti-Mb antibodies in vitro if they received F gamma G-specific T cell help. However, (high-responder X low responder) F1 T cells from Mb-immune mice did not help these primed low responder (H-2k or H-2b) B cells in vitro, even in the presence of various numbers of F1 APC that were demonstrated to be component to reconstitute the response of spleen cells depleted by APC. Similar results were obtained with B6 leads to B6D2F1 radiation bone marrow chimeras. Genotypic low responder (H-2b) T cells from these mice helped Mb-primed B6D2F1B cells plus APC, but did not help syngeneic chimeric H-2b B cells, even in the presence of F1 APC. In contrast, we could not detect any Ir restriction on APC function during these in vitro secondary responses. Moreover, in the preceding paper, we found that low responder mice neonatally tolerized to higher responder H-2 had competent Mb-specific helper T cells capable of helping high responder but not low responder B cells and APC. Therefore, although function Mb-specific T cells and B cells both exist in low responder mice, the Ir gene defect is a manifestation of the failure of syngeneic collaboration between these two cell types. This genetic restriction on the interaction between T cells and B cells is consistent with the additional new finding that Lyb-5-negative B cells are a major participant in ths vitro secondary response because it is this Lyb-5-negative subpopulation of B cells that have recently been shown to require genetically restricted help. The Ir gene defect behaves operationally as a failure of low responder B cells to receive help from any source of Mb-specific T cells either high responder, low responder, or F1. The possible additional role of T cell-APC interactions, either during primary immunization in vivo or in the secondary culture is discussed.

scholarly journals MATURATION OF THE HUMORAL IMMUNE RESPONSE IN MICE

1974 ◽  
Vol 139 (2) ◽  
pp. 249-263 ◽  
Author(s):  
Patricia G. Spear ◽  
Gerald M. Edelman
Keyword(s):  
T Cell ◽  
B Cells ◽  
Cell Function ◽  
Spleen Cells ◽  
Con A ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Humoral Antibodies

In spite of the prenatal appearance of immunoglobulin-bearing lymphocytes and θ-positive lymphocytes in the spleens of Swiss-L mice, these mice are not able to produce detectable levels of humoral antibodies in response to antigen until after 1 wk of age. Adult levels of response are not achieved until 4–8 wk of age. In the presence of bacterial lipopolysaccharides, which can substitute for or enhance T-cell function, the B cells from young Swiss-L mice were found to be indistinguishable in function from adult B cells, both with respect to the numbers of plaque-forming cells (PFC) produced in vitro in response to antigen and with respect to the kinetics of PFC induction. The spleen cells from young Swiss-L mice are significantly less sensitive than adult spleen cells, however, to stimulation by the T cell mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA). Very few Con A-responsive cells could be detected at birth but the numbers increased sharply with age until 3 wk after birth. On the other hand, PHA-responsive cells could not be detected in the spleen until about 3 wk of age. The latter cells were found to respond also to Con A, but at a lower dose (1 µg/ml) than that required for the bulk of the Con A-responsive cells (3 µg/ml). The cells that respond both to PHA and to Con A appear in the spleen at about the time that Swiss-L mice acquire the ability to produce humoral antibodies, and these cells can be depleted from the spleen by the in vivo administration of antithymocyte serum. The development of humoral immune responses in these mice therefore appears to be correlated with the appearance of recirculating T lymphocytes that are responsive both to PHA and to Con A.

scholarly journals The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1134
Author(s):  
Won-Ju Kim ◽  
Gil-Ran Kim ◽  
Hyun-Jung Cho ◽  
Je-Min Choi
Keyword(s):  
Drug Delivery ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Autoimmune Encephalomyelitis ◽  
Optimal Sequence ◽  
T Cell Function

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood–brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

Effective Prevention of Acute Graft-Verses-Host Disease by Targeting PKC Alpha and PKC Theta in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1898-1898
Author(s):  
Kelley M.K. Haarberg ◽  
Crystina Bronk ◽  
Dapeng Wang ◽  
Amer Beg ◽  
Xue-Zhong Yu
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Acute Gvhd ◽  
T Cell Signaling ◽  
Effector Cells ◽  
T Effector Cells ◽  
Immunologic Synapse

Abstract Abstract 1898 Protein kinase C theta (PKCθ), a T cell signaling molecule, has been implicated as a therapeutic target for several autoimmune diseases as well as graft-versus-host disease (GVHD). PKCθ plays a vital role in stabilization of the immunologic synapse between T effector cells and antigen presenting cells (APC), but has been shown to be excluded from the immunologic synapse in T regulatory cells (T reg). PKCθ inhibition reduces the alloreactivity of donor T cells responsible for induction of GVHD while preserving graft-versus-leukemia (GVL) responses. The roles of PKCθ and the potential compensatory alpha isoform (PKCα) are not clearly defined with regard to alloresponses or T cell mediated responses in GVHD. In this context, we measured PKCθ and PKCα/θ gene deficient T cell activation upon TCR-ligation in vitro using [3H]-TdR incorporation and CSFE labeling assays. T cells from PKCθ and PKCα/θ gene deficient donor mice were utilized in vivo in a pre-clinical allogenic murine model of myeloablative bone marrow transplantation (BMT). The development of GVHD was monitored in recipient mice with or without injection of A20-luciferase cells to observe the progression of GVL in vivo. Combined blockade of PKCα and PKCθ causes a significant decrease in T cell proliferation compared to blocking PKCθ alone in vitro. Deficiency in PKCα and PKCθ had no effect on immune reconstitution following irradiation and BMT in vivo. Even with a high transplant load of 5×106 CD4+ and CD8+ T cells, PKCα/θ deficient (PKCα/θ−/−) T cells failed to induce acute GVHD. Our data suggest that the ability of double deficient T cells to induce GVHD was further reduced than PKCθ-deficient T cells. Additionally, a greater number and percentage of B220+ B cells and FoxP3+ T regs were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type (WT) or PKCθ−/− T cell recipients. Fewer CD4+ or CD8+ T effector cells were isolated from the spleens of PKCα/θ−/− T cell recipient mice 120 after BMT than were isolated from wild type or PKCθ−/− T cell recipients. Importantly, the activity of B cells isolated from PKCα/θ−/− T cell recipient mice 120 after BMT was greater on a per cell basis, while the activity of T effector cells isolated from these mice was greatly reduced compared to WT or PKCθ−/− T cell recipients. While not absent, GVL was reduced in PKCα/θ−/− T cell recipient mice when compared to WT or PKCθ−/− T cell recipients. This work demonstrates the requirement of PKCα and θ for optimal activation and function of T cells in vitro. These experiments highlight a potential compensatory role for PKCα in the absence of PKCθ in T cell signaling and activation. Combined deficiency of PKCα and θ prevents induction of acute GVHD while improving the maintenance of splenic cellularity in PKCα/θ T cell recipient mice. Additionally, PKCα/θ dual deficient T cell transplant shifts the splenic balance toward a greater number and percentage of T reg and B cells and away from T effector cells following BMT. The reduced and sub-optimally active T effector cells isolated from PKCα/θ−/− T cell recipient mice in combination with reduced GVL stresses the importance of PKCα and θ molecules and their roles in T cell activity in the context of both GVHD and GVL. Dual deficiency of PKCα/θ is associated with a decline of T effector function that is optimal for the amelioration of GVHD, but is perhaps too reduced to substantially maintain effective GVL. Modulation of PKCα and θ signaling presents a valid avenue of investigation as a therapeutic option for GVHD. Disclosures: No relevant conflicts of interest to declare.

CD3 limits the efficacy of TCR gene therapy in vivo

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3528-3537 ◽  
Author(s):  
Maryam Ahmadi ◽  
Judith W. King ◽  
Shao-An Xue ◽  
Cécile Voisine ◽  
Angelika Holler ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Cell Receptor ◽  
Surface Expression ◽  
T Cell Function ◽  
Engineered T Cells ◽  
Rate Limiting

Abstract The function of T-cell receptor (TCR) gene modified T cells is dependent on efficient surface expression of the introduced TCR α/β heterodimer. We tested whether endogenous CD3 chains are rate-limiting for TCR expression and antigen-specific T-cell function. We show that co-transfer of CD3 and TCR genes into primary murine T cells enhanced TCR expression and antigen-specific T-cell function in vitro. Peptide titration experiments showed that T cells expressing introduced CD3 and TCR genes recognized lower concentration of antigen than T cells expressing TCR only. In vivo imaging revealed that TCR+CD3 gene modified T cells infiltrated tumors faster and in larger numbers, which resulted in more rapid tumor elimination compared with T cells modified by TCR only. After tumor clearance, TCR+CD3 engineered T cells persisted in larger numbers than TCR-only T cells and mounted a more effective memory response when rechallenged with antigen. The data demonstrate that provision of additional CD3 molecules is an effective strategy to enhance the avidity, anti-tumor activity and functional memory formation of TCR gene modified T cells in vivo.

scholarly journals Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility

2017 ◽  
Author(s):  
Tobias X. Dong ◽  
Shivashankar Othy ◽  
Milton L. Greenberg ◽  
Amit Jairaman ◽  
Chijioke Akunwafo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Motility ◽  
Immune Surveillance ◽  
Cellular Motility ◽  
Average Cell ◽  
Cell Functions ◽  
Human T Cells

AbstractCa2+ influx through Orai1 channels is crucial for several T cell functions, but a role in regulating basal cellular motility has not been described. Here we show that inhibition of Orai1 channel activity increases average cell velocities by reducing the frequency of pauses in human T cells migrating through confined spaces, even in the absence of extrinsic cell contacts or antigen recognition. Utilizing a novel ratiometric genetically encoded cytosolic Ca2+ indicator, Salsa6f, which permits real-time monitoring of cytosolic Ca2+ along with cell motility, we show that spontaneous pauses during T cell motility in vitro and in vivo coincide with episodes of cytosolic Ca2+ signaling. Furthermore, lymph node T cells exhibited two types of spontaneous Ca2+ transients: short-duration “sparkles” and longer duration global signals. Our results demonstrate that spontaneous and self-peptide MHC-dependent activation of Orai1 ensures random walk behavior in T cells to optimize immune surveillance.

scholarly journals PRMT5 Is Upregulated in Activated T Cells and Is a Novel Therapeutic Target for Acute Gvhd

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2034-2034
Author(s):  
Parvathi Ranganathan ◽  
Katiri Snyder ◽  
Nina Zizter ◽  
Hannah K. Choe ◽  
Robert A Baiocchi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Western Blot ◽  
Cell Function ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
And Function

Abstract Introduction: Acute graft-versus-host disease (aGVHD), a T cell-mediated immunological disorder is the leading cause of non-relapse mortality in patients receiving allogeneic bone marrow transplants. Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (me2s) of arginine (R) residues on histones (primarily H3R8 and H3R4) and other proteins. PRMT5 is overexpressed in many leukemias and lymphomas, and epigenetic changes driven by PRMT5 lead to repression of tumor suppressors and promote growth and survival of cancer cells. Recently it was shown that T cells are sensitive to R-methylation and PRMT5 promotes activation of memory T helper cells. Here we investigate: 1) mechanisms by which PRMT5 regulates T cell function; and 2) PRMT5 inhibition as a therapeutic strategy for aGVHD. Materials and Methods: Splenic T cells were isolated from lethally irradiated B6D2F1 mice that received either T cell depleted bone marrow (TCD-BM) or TCD-BM with C57/BL6 (B6) allogeneic splenocytes on day 21 post-transplant. In vitro activation of B6 T cells was achieved with CD3/CD28 Dynabeads or co-culture with allogeneic BM-derived dendritic cells. PRMT5 expression (RT-PCR, western blot) and function (H3R8me2s western blot) were evaluated. PRT220, a novel inhibitor of PRMT5, was used to evaluate PRMT5 inhibition on T cell function in vitro and in vivo. We assessed T cell proliferation (Cell Trace Violet, Ki67), apoptosis (Annexin V), cytokine secretion (ELISA, flow cytometry), cell cycle (PI incorporation), and cell signaling (western blot). Lethally irradiated F1 recipients received TCD-BM only (10x106 cells) or TCD-BM + B6 splenocytes (20 x 106). Recipients of allogeneic splenocytes were treated with PRT220 (2mg/kg) or vehicle by oral gavage once weekly starting day 7 post-transplant. Mice were monitored for survival and clinical aGVHD scores. Results: PRMT5 expression and function is upregulated following T cell activation. Inhibition of PRMT5 reduces T cell proliferation and IFN-g secretion. PRMT5 inhibition in CD3/CD28 stimulated T cells results in disruption of multiple histone epigenetic marks, cell-cycle progression (via G1 arrest) and perturbation of ERK-MAPK signaling cascades. Finally, administration of PRT220 resulted in significantly prolonging the survival of allo-transplanted recipient mice (median survival, PRT220 vs. vehicle, 36.5 vs. 26 days, p=0.01). PRT220-treated recipients also exhibited significant lower aGVHD clinical (p<0.05), pathological scores (p<0.05) and lower serum TNF-a (p<0.05) and IFN-g (p<0.05) than vehicle-treated recipients. Conclusions: PRMT5 expression and function are upregulated in activated T cells. Inhibition of PRMT5 function using a novel and specific small-molecule inhibitor, PRT220, down-regulates T cells proliferative and effector response, induces cell-cycle arrest and perturbs signaling pathways. PRT220 shows potent biological activity in vivo by reducing aGVHD clinical severity and significantly prolonging survival in mouse models of aGVHD. Therefore, PRMT5 is a novel and druggable target for aGVHD. Disclosures No relevant conflicts of interest to declare.

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