Abstract 20: Apolipoprotein A-I Influences Regulatory T Cell Development and Proliferation in Homeostasis and Atherogenesis

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Dalia E Gaddis ◽  
Amy Wu ◽  
Debbi Yoakum ◽  
Mary Sorci-Thomas ◽  
Catherine C Hedrick
Keyword(s):  
T Cell ◽  
Atherogenic Diet ◽  
Cholesterol Homeostasis ◽  
Major Protein ◽  
Apolipoprotein A ◽  
Brdu Labeling ◽  
And Function ◽  
Treg Development

Apolipoprotein A-I (ApoAI) is the major protein component of HDL. HDL ApoAI is involved in the efflux of cholesterol from cells to maintain cellular cholesterol homeostasis. ApoAI also has anti-inflammatory properties. We previously showed that injecting mice fed an atherogenic diet with ApoAI decreased the number of activated CD4 lymphocytes. Since T regulatory lymphocytes (Treg) play a major role in inhibiting the immune response during atherosclerosis development, we wanted to determine if ApoAI influences Treg development, hypothesizing that ApoAI enhances Treg development. To test this hypothesis, we compared the numbers of Treg in ApoAI-/- mice to B6 mice, and found a 50% decrease in the numbers of Treg in the periaortic LNs (PaLN) of ApoAI-/- mice. BrdU labeling studies showed that ApoAI-/- Treg had a significant 30% reduction in proliferation, suggesting that in the absence of ApoAI and normal cholesterol homeostasis, Tregs have defective proliferation. Functionally, we discovered that ApoAI-/- Treg were significantly less suppressive than B6 Treg in reducing CD4 effector T cell proliferation, suggesting that ApoAI plays a role in both the development and function of Tregs. To determine if the addition of exogenous lipid-free ApoAI could rescue and promote Treg differentiation in ApoAI-/- mice, ApoAI-/- naïve T cells were incubated in vitro with TGFβ and exogenous ApoAI. Addition of ApoAI significantly increased development of naïve ApoA1-/- lymphocytes into Treg. To verify these results in vivo, we fed a novel Treg lineage tracker mouse (LT), Foxp3-YFP-Cre-Rosa26-RFP-ApoE-/- mice a western diet for 15 weeks and administered subcutaneous injections of ApoAI for the last 9 weeks of diet. These mice allow us to identify current functional Tregs and any exTregs that have lost active Treg function in vivo. We found that LT mice treated with ApoAI had a 37% decrease in exTregs and a concomitant 33% increase in current functional Tregs in the aorta. This was accompanied by decreased IFNγ and IL-17 production in PaLN, further confirming our in vitro findings that ApoAI promotes Treg development and function. In conclusion, we have identified a novel role for ApoAI by enhancing Treg development, emphasizing the immune properties of ApoAI for atheroprotection.

scholarly journals The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

2005 ◽  
Vol 25 (23) ◽  
pp. 10533-10542 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Christian Schmedt ◽  
Masato Okada ◽  
Alexander Tarakhovsky
Keyword(s):  
T Cell ◽  
Adaptor Protein ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Thymic Development ◽  
Cell Functions ◽  
Carboxy Terminal ◽  
And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

scholarly journals 173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A185-A185
Author(s):  
Michelle Fleury ◽  
Derrick McCarthy ◽  
Holly Horton ◽  
Courtney Anderson ◽  
Amy Watt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
T Cell Receptor ◽  
Cytokine Production ◽  
Cell Receptor ◽  
Great Promise ◽  
And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

scholarly journals Tumor-Related Exosomes Contribute to Tumor-Promoting Microenvironment: An Immunological Perspective

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Wuzhen Chen ◽  
Jingxin Jiang ◽  
Wenjie Xia ◽  
Jian Huang
Keyword(s):  
T Cell ◽  
Cell Activation ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Suppressor Cells ◽  
Effector T Cell ◽  
T Cell Apoptosis ◽  
And Function

Exosomes are a kind of cell-released membrane-form structures which contain proteins, lipids, and nucleic acids. These vesicular organelles play a key role in intercellular communication. Numerous experiments demonstrated that tumor-related exosomes (TEXs) can induce immune surveillance in the microenvironment in vivo and in vitro. They can interfere with the maturation of DC cells, impair NK cell activation, induce myeloid-derived suppressor cells, and educate macrophages into protumor phenotype. They can also selectively induce effector T cell apoptosis via Fas/FasL interaction and enhance regulatory T cell proliferation and function by releasing TGF-β. In this review, we focus on the TEX-induced immunosuppression and microenvironment change. Based on the truth that TEXs play crucial roles in suppressing the immune system, studies on modification of exosomes as immunotherapy strategies will also be discussed.

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.

scholarly journals Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ioana Sandu ◽  
Dario Cerletti ◽  
Manfred Claassen ◽  
Annette Oxenius
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Viral Infections ◽  
Cell Receptor ◽  
Target Cells ◽  
Tcr Signaling ◽  
And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

IL15, but Not IL2, Supports Long-Term Survival and Function of Human and Macaque Antigen-Specific CD8+ T Cell Clones.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3237-3237
Author(s):  
Carolina S. Berger ◽  
Michael Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cell ◽  
Term Survival ◽  
Long Term Survival ◽  
Cd8 T Cell Memory ◽  
And Function

Abstract The adoptive transfer of antigen-specific CD8+ cytotoxic T lymphocyte (CTL) clones that have been isolated and expanded in vitro is a promising treatment modality for both human malignancies and infections. However, establishing immunity of sufficient magnitude and persistence for sustained efficacy is a limitation of this approach. Recent studies have identified a critical role for cytokine signaling including that mediated by IL15 in the establishment and maintenance of CD8+ T cell memory, suggesting that protocols for generating and transferring antigen-specific T cells might be improved. Interleukin-2 (IL2) is the T cell growth factor that has been widely used in vitro and in vivo for promoting T cell proliferation and persistence, but prolonged exposure of T cells to IL2 can enhance susceptibility to cell death and limit CD8+ memory T cell survival. IL15 is a novel cytokine that shares some activities with IL2 such as the induction of T cell proliferation, but exerts contrasting effects on the homeostasis of CD8+ T cell memory in experimental models. Here, we study the utility of IL15 to enhance the long-term survival and function of human and macaque antigen-specific CD8+ CTL clones in vitro. Human and macaque CD8+ CTL clones reactive against CMV were isolated by limiting dilution, expanded over 14 days in the presence of IL2 or IL15 (1–10 ng/ml), and then rested for &gt;4 weeks in media alone and with IL2 or IL15 at 0.01–10 ng/ml. Surviving T cells were enumerated at intervals, monitored for cell surface phenotype, and assayed for cytotoxicity by chromium release assay. CTL expanded in IL2 or IL15 proliferated equivalently over 14 days with a median of 1100 and 1400 fold increase in number, displayed surface markers consistent with an effector memory phenotype (CD45RA−CD62L−CCR7−CD28−), and showed comparable cytotoxicity (n=4). However, exposure after 14 days to IL15 at doses as little as 0.05-0.1 ng/ml greatly enhanced the survival of the CD8+ CTL as determined by Annexin V staining. By contrast, cells cultured without cytokines or with IL2 declined &gt;80% in number over 3 or 11 days, respectively. Of note, IL15 at higher doses (&gt;0.5 ng/ml), but not IL2, efficiently promoted sustained cell growth illustrated by labeling cells with CFSE. Cells cultured with IL15 displayed 1.5-fold increased expression of antiapoptotic molecules such as Bcl-xL and Bcl-2 over those plated in IL2 (n=4), indicating IL15 mediated its effects at least in part by preventing apoptosis. Of note, the cytotoxicity of CTL rested in IL2 was markedly reduced (&gt;60%, n=3), while the presence of IL15 permitted for sustained CTL function and expansion after restimulation. The responses of human and macaque CTL clones to IL15 were equivalent suggesting in vivo studies of T cell transfer in macaques may be predictive of results in humans. We have constructed retroviral vectors encoding intracytoplasmic truncated macaque CD34 or CD19 genes that could serve as nonimmunogenic selectable marker to track macaque T cells after transfer. Macaque T cells were efficiently transduced to express CD34t and CD19t (&gt;50%), and enriched to high purity by immunomagnetic selection. Studies to examine the safety and utility of IL15 on the survival of adoptively transferred CTL in macaques are in progress. Collectively, our data support that novel cytokines such as IL15 may prove useful to augment the long-term survival and effector function of ex vivo expanded antigen-specific CD8+ CTL clones after transfer.

scholarly journals Myeloid-derived suppressor cell subtypes differentially influence T-cell function, T-helper subset differentiation, and clinical course in CLL

Leukemia ◽  
2021 ◽  
Author(s):  
Gerardo Ferrer ◽  
Byeongho Jung ◽  
Pui Yan Chiu ◽  
Rukhsana Aslam ◽  
Florencia Palacios ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Function ◽  
Clinical Course ◽  
Suppressor Cells ◽  
Suppressor Cell ◽  
Cancer Pathogenesis ◽  
Myeloid Derived Suppressor Cell ◽  
And Function

AbstractCancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

scholarly journals Repositioning Azelnidipine as a Dual Inhibitor Targeting CD47/SIRPα and TIGIT/PVR Pathways for Cancer Immuno-Therapy

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 706
Author(s):  
Xiuman Zhou ◽  
Ling Jiao ◽  
Yuzhen Qian ◽  
Qingyu Dong ◽  
Yixuan Sun ◽  
...  
Keyword(s):  
T Cell ◽  
Cancer Immunotherapy ◽  
Cd8 T Cell ◽  
Dependent Manner ◽  
Dual Inhibitor ◽  
Macrophage Phagocytosis ◽  
Hypertensive Drug ◽  
And Function

Strategies boosting both innate and adaptive immunity have great application prospects in cancer immunotherapy. Antibodies dual blocking the innate checkpoint CD47 and adaptive checkpoint PD-L1 or TIGIT could achieve durable anti-tumor effects. However, a small molecule dual blockade of CD47/SIRPα and TIGIT/PVR pathways has not been investigated. Here, an elevated expression of CD47 and PVR was observed in tumor tissues and cell lines analyzed with the GEO datasets and by flow cytometry, respectively. Compounds approved by the FDA were screened with the software MOE by docking to the potential binding pockets of SIRPα and PVR identified with the corresponding structural analysis. The candidate compounds were screened by blocking and MST binding assays. Azelnidipine was found to dual block CD47/SIRPα and TIGIT/PVR pathways by co-targeting SIRPα and PVR. In vitro, azelnidipine could enhance the macrophage phagocytosis when co-cultured with tumor cells. In vivo, azelnidipine alone or combined with irradiation could significantly inhibit the growth of MC38 tumors. Azelnidipine also significantly inhibits the growth of CT26 tumors, by enhancing the infiltration and function of CD8+ T cell in tumor and systematic immune response in the tumor-draining lymph node and spleen in a CD8+ T cell dependent manner. Our research suggests that the anti-hypertensive drug azelnidipine could be repositioned for cancer immunotherapy.

scholarly journals Fever supports CD8+ effector T cell responses by promoting mitochondrial translation

2021 ◽  
Vol 118 (25) ◽  
pp. e2023752118
Author(s):  
David O’Sullivan ◽  
Michal A. Stanczak ◽  
Matteo Villa ◽  
Franziska M. Uhl ◽  
Mauro Corrado ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Metabolic Activity ◽  
Cell Metabolism ◽  
Transcriptional Profiling ◽  
Mitochondrial Translation ◽  
In Vivo Models ◽  
And Function

Fever can provide a survival advantage during infection. Metabolic processes are sensitive to environmental conditions, but the effect of fever on T cell metabolism is not well characterized. We show that in activated CD8+ T cells, exposure to febrile temperature (39 °C) augmented metabolic activity and T cell effector functions, despite having a limited effect on proliferation or activation marker expression. Transcriptional profiling revealed an up-regulation of mitochondrial pathways, which was consistent with increased mass and metabolism observed in T cells exposed to 39 °C. Through in vitro and in vivo models, we determined that mitochondrial translation is integral to the enhanced metabolic activity and function of CD8+ T cells exposed to febrile temperature. Transiently exposing donor lymphocytes to 39 °C prior to infusion in a myeloid leukemia mouse model conferred enhanced therapeutic efficacy, raising the possibility that exposure of T cells to febrile temperatures could have clinical potential.

TNF-Alpha Priming Enhances CD4+ FoxP3+ Regulatory T Cell Suppressive Function in GvHD Prevention and Treatment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1885-1885
Author(s):  
Antonio Pierini ◽  
Caitlin Moffett ◽  
Dominik Schneidawind ◽  
Jeanette Baker ◽  
Hidekazu Nishikii ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Peripheral Blood ◽  
Activation Markers ◽  
Suppressive Function ◽  
Prevention And Treatment ◽  
And Function

Abstract CD4+ CD25+ FoxP3+ regulatory T cells (Treg) have been shown to effectively prevent graft versus host disease (GvHD) when adoptively transferred in murine models of hematopoietic cell transplantation (HCT) and phase I/II clinical trials. Critical limitations to the clinical application of Treg are the paucity of cells and limited knowledge of the mechanism(s) of in vivo function. In physiologic conditions Treg regulate immune responses during inflammation. We hypothesized that inflammatory conditions in GvHD modify Treg characteristics and function. To test this hypothesis, we primed Treg with irradiated (3000 cGy) peripheral blood from acute GvHD (aGvHD) affected mice for 20-24 hours and then transferred these cells in a mouse model of GvHD where allogeneic T cell depleted bone marrow (TCD BM) from C57BL/6 mice was transplanted into lethally irradiated (8 Gy) BALB/c recipients together with 7.5x105 to 1x106 /animal donor derived conventional CD4+ and CD8+ T cells (Tcon). C57BL/6 Treg primed with irradiated aGvHD peripheral blood were injected at day 0 after HCT for preventing GvHD or at day +7 or +8 as GvHD treatment. Their adoptive transfer resulted in improved survival in comparison to unprimed natural occurring Treg when used for both GvHD prevention (p=0.01) and treatment (p=0.04). Moreover treatment with irradiated aGvHD peripheral blood-primed Treg did not impact graft versus tumor effects in a mouse model of T cell mediated tumor killing. BLI demonstrated that injected allogeneic Tcon completely cleared previously infused luc+ A20 tumor cells even in the presence of primed Treg (primed Treg + Tcon + A20 vs A20 alone p<0.001). Irradiated aGvHD peripheral blood-primed Treg express increased levels of activation markers with suppressive function such as CTLA4 (p<0.001) and LAG3 (p<0.05) in comparison to unprimed Treg in vitro. We also found that Treg primed with irradiated cells of aGvHD affected animals after removing the serum did not enhance the expression of the same markers (p>0.05) demonstrating that serum from aGvHD animals is required for Treg priming and function. We further tested the ability of several inflammatory cytokines that are normally secreted during GvHD such as IFN-γ, IL-6, IL-12 and TNFα to induce similar in vitro Treg activation. We found that TNFɑ selectively activated Treg without impacting CD4+ FoxP3- T cells. TNFɑ-primed Treg have increased expression of activation markers such as CD69 (p<0.0001), CD25 (p<0.0001), and LAG3 (p=0.0002), produce a greater amount of suppressive cytokines such as IL-10 (p=0.03) and TGF-β (p=0.02), and enhance the expression of homing markers such as CD62L (p=0.005) that are required for in vivo function. TNFɑ-primed Treg had increased ability to proliferate (p=0.02) and, at the same time, to suppress Tcon proliferation (p=0.04) in a mixed lymphocyte reaction against irradiated allogeneic splenocytes, while, on the contrary, TNFɑ-primed Tcon had reduced ability to proliferate in similar conditions in comparison to unprimed Tcon (p=0.0004). To test the effect of TNFɑ priming on in vivo Tcon proliferation we injected TNFɑ-primed and unprimed luc+ Tcon in allogeneic BALB/c Rag2-/- γ-chain-/- immune deficient animals that were sublethally irradiated (400 cGy). BLI at day +7 after Tcon injection revealed reduced TNFɑ-primed Tcon in vivo proliferation (p=0.01) that resulted in milder GvHD symptoms (p=0.02). Finally, in a GvHD prevention mouse model TNFɑ-primed Treg infused at 1:10 Treg/Tcon ratio resulted in improved animal survival as compared to unprimed Treg (p=0.02), demonstrating enhanced efficacy of TNFɑ priming in the in vivo function of Treg. In summary, our study demonstrates that Treg respond to TNFɑ acquiring an activated status resulting in increased function. As TNFɑ is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore TNFɑ priming of Treg in vitro provides a new tool to optimize Treg cellular therapies also allowing for the use of a reduced cell number for GvHD prevention and treatment. Disclosures No relevant conflicts of interest to declare.

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