IL-23 induces regulatory T cell plasticity with implications for inflammatory skin diseases

AbstractFoxp3+ regulatory T cells (Tregs) represent a major fraction of skin resident T cells. Although normally protective, Tregs have been shown to produce pro-inflammatory cytokines in human diseases, including psoriasis. A significant hurdle in the Treg field has been the identification, or development, of model systems to study this Treg plasticity. To overcome this gap, we analyzed skin resident Tregs in a mouse model of IL-23 mediated psoriasiform dermatitis. Our results demonstrate that IL-23 drove the accumulation of Tregs; including a subpopulation that co-expressed RORγt and produced IL-17A. Genesis of this population was attenuated by a RORγt inverse agonist compound and clinically relevant therapeutics. In vitro, IL-23 drove the generation of CD4+Foxp3+RORγt+IL-17A+ cells from Treg cells. Collectively, our data shows that IL-23 drives Treg plasticity by inducing a population of CD4+Foxp3+RORγt+IL-17A+ cells that could play a role in the disease pathogenesis. Through this work, we define an in vitro system and a pre-clinical in vivo mouse model that can be used to further study Treg homeostasis and plasticity in the context of psoriasis.

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T-cell–intrinsic Tif1α/Trim24 regulates IL-1R expression on TH2 cells and TH2 cell-mediated airway allergy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522287113 ◽

2016 ◽

Vol 113 (5) ◽

pp. E568-E576 ◽

Cited By ~ 10

Author(s):

Jimena Perez-Lloret ◽

Isobel S. Okoye ◽

Riccardo Guidi ◽

Yashaswini Kannan ◽

Stephanie M. Coomes ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Allergic Diseases ◽

Treg Cells ◽

Th2 Cells ◽

T Helper 2 ◽

Cytokines And Chemokines ◽

Th2 Cell

There is a paucity of new therapeutic targets to control allergic reactions and forestall the rising trend of allergic diseases. Although a variety of immune cells contribute to allergy, cytokine-secreting αβ+CD4+ T-helper 2 (TH2) cells orchestrate the type-2–driven immune response in a large proportion of atopic asthmatics. To identify previously unidentified putative targets in pathogenic TH2 cells, we performed in silico analyses of recently published transcriptional data from a wide variety of pathogenic TH cells [Okoye IS, et al. (2014) Proc Natl Acad Sci USA 111(30):E3081–E3090] and identified that transcription intermediary factor 1 regulator-alpha (Tif1α)/tripartite motif-containing 24 (Trim24) was predicted to be active in house dust mite (HDM)- and helminth-elicited Il4gfp+αβ+CD4+ TH2 cells but not in TH1, TH17, or Treg cells. Testing this prediction, we restricted Trim24 deficiency to T cells by using a mixed bone marrow chimera system and found that T-cell–intrinsic Trim24 is essential for HDM-mediated airway allergy and antihelminth immunity. Mechanistically, HDM-elicited Trim24−/− T cells have reduced expression of many TH2 cytokines and chemokines and were predicted to have compromised IL-1–regulated signaling. Following this prediction, we found that Trim24−/− T cells have reduced IL-1 receptor (IL-1R) expression, are refractory to IL-1β–mediated activation in vitro and in vivo, and fail to respond to IL-1β–exacerbated airway allergy. Collectively, these data identify a previously unappreciated Trim24-dependent requirement for IL-1R expression on TH2 cells and an important nonredundant role for T-cell–intrinsic Trim24 in TH2-mediated allergy and antihelminth immunity.

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Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+ regulatory T-cells

Molecular Biology of the Cell ◽

10.1091/mbc.e14-08-1323 ◽

2015 ◽

Vol 26 (15) ◽

pp. 2845-2857 ◽

Cited By ~ 51

Author(s):

Magdalena Walecki ◽

Florian Eisel ◽

Jörg Klug ◽

Nelli Baal ◽

Agnieszka Paradowska-Dogan ◽

...

Keyword(s):

T Cells ◽

Androgen Receptor ◽

Treg Cell ◽

Foxp3 Expression ◽

Treg Cells ◽

Strong Increase ◽

Histone H4 ◽

And Function

CD4+CD25+Foxp3+ regulatory T (Treg) cells are able to inhibit proliferation and cytokine production in effector T-cells and play a major role in immune responses and prevention of autoimmune disease. A master regulator of Treg cell development and function is the transcription factor Foxp3. Several cytokines, such as TGF-β and IL-2, are known to regulate Foxp3 expression as well as methylation of the Foxp3 locus. We demonstrated previously that testosterone treatment induces a strong increase in the Treg cell population both in vivo and in vitro. Therefore we sought to investigate the direct effect of androgens on expression and regulation of Foxp3. We show a significant androgen-dependent increase of Foxp3 expression in human T-cells from women in the ovulatory phase of the menstrual cycle but not from men and identify a functional androgen response element within the Foxp3 locus. Binding of androgen receptor leads to changes in the acetylation status of histone H4, whereas methylation of defined CpG regions in the Foxp3 gene is unaffected. Our results provide novel evidence for a modulatory role of androgens in the differentiation of Treg cells.

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Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽

10.1182/blood.v106.11.5256.5256 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5256-5256

Author(s):

Doug Cipkala ◽

Kelly McQuown ◽

Lindsay Hendey ◽

Michael Boyer

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Mouse Model ◽

Scid Mouse ◽

In Vitro Cytotoxicity ◽

Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p>0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p>0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p<0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

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Inhibition Of Cdk2 Promotes The Generation Of Inducible CD8+ T Regulatory Cells By Modulating The Epigenetic Regulator EZH2

Blood ◽

10.1182/blood.v122.21.138.138 ◽

2013 ◽

Vol 122 (21) ◽

pp. 138-138

Author(s):

Lequn Li ◽

Nikolaos Patsoukis ◽

Anoma Nellore ◽

Vassiliki A. Boussiotis

Keyword(s):

T Cells ◽

T Cell ◽

Fold Increase ◽

Treg Cells ◽

Foxp3 Mrna ◽

Alloreactive T Cells ◽

Epigenetic Regulator ◽

Treg Differentiation

Abstract Graft versus host disease (GvHD) remains the main cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation. In spite of the intense research efforts, control of GvHD remains incomplete and novel therapeutic approaches are required. Cdk2 has a central role in cell cycle re-entry of mature T lymphocytes and inhibition of Cdk2 is mandatory for induction of T cell anergy in vitro and tolerance in vivo. While Cdk2 is essential for expansion of activated T cells, it is not critical for survival of resting lymphocytes, hematopoiesis or thymocyte development. These properties make Cdk2 an attractive target for control of GvHD. To determine the effects of Cdk2 inhibition on T cell alloresponses in vivo, we used the B6D2F1 mouse model of allogeneic BMT and two different Cdk2 inhibitors, CYC202 (IC50=0.1 uM) and CYC205 (IC50=1 nM). Lethally irradiated B6D2F1(Kd) recipients were infused with bone marrow from C57BL/6(Kb) donors with (BMT) or without splenocytes (BM) and were subsequently treated with each Cdk2 inhibitor for three weeks. Treatment was administered daily during week 1, every other day on week 2, and twice a week on week 3. Effects of treatment on GvHD were assessed by body weight and survival during a 70-day period. Although BMT recipients treated with Cdk2 inhibitor displayed a transient initial weight loss, subsequently regained weight to levels comparable to control BM recipients. Furthermore, treated BMT recipient groups displayed significantly delayed GvHD mortality (p=0.0054). Recently, it was determined that inducible CD8+ Treg cells, have a central role in mediating protection from GvHD. Some immunosuppressive drugs have detrimental effects on Treg whereas others spare these cells or may even be beneficial to their proportional increase. To examine whether Cdk2 inhibitors induced Treg cells, we used GFP- T cells from Foxp3.GFP-KI mice (C57BL/6 background) as a source of T cells during BMT. Assessment of peripheral blood lymphocytes, splenocytes, peripheral lymph nodes and intestinal lymphoid cells (ILC) in BMT recipients revealed no differences in CD4+GFP+ Treg between treated and control groups. In contrast, the treated group displayed an increase of CD8+GFP+ Treg cells in these cell populations, predominantly ILC, which displayed a 5-fold increase of CD8+ Treg (p=0.05). To further investigate whether Cdk2 inhibitors had a selective effect on CD8+ Treg differentiation, we isolated CD4+GFP- and CD8+GFP- T cells from Foxp3.GFP-KI mice and subjected them to in vitro Treg polarizing with or without Cdk2 inhibitors. Inhibition of Cdk2 had almost no effect on CD4+GFP+ cells but induced a 2-4 fold increase of CD8+GFP+ cells. To determine whether Cdk2 inhibition induced its effect on CD8+ Treg differentiation by reducing the threshold of TGF-β-mediated signaling, we cultured CD8+GFP- cells with stable concentrations of Cdk2 inhibitors and decreasing concentrations of TGF-β. Cdk2 inhibition induced CD8+ Treg differentiation in the presence of TGF-β concentrations that failed to induce any significant numbers of CD8+ Treg cells when used alone. Expression of FOX family genes is regulated by transcriptional and epigenetic mechanisms. A critical epigenetic regulator of FOX transcription factors in cancer cells is the Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), which promotes histone H3 lysine 27 trimethylation (H3K27me3) and induces epigenetic gene silencing. Cdk1 and Cdk2 phosphorylate EZH2 at Thr350 in an evolutionarily conserved motif. Phosphorylation of Thr350 is important for EZH2 recruitment and maintenance of H3K27me3 levels at EZH2-target loci. We examined whether EZH2 becomes phosphorylated in CD8+ T cells and whether Cdk2 inhibition might affect this event. Upon polarizing CD8+ T cell culture, EZH2 displayed robust phosphorylation on Thr350, which was blocked by Cdk2 inhibition. This event temporally coincided with a 44-fold increase in Foxp3 mRNA expression compared to base line levels in control T cells. These results reveal an unexpected mechanism via which Cdk2 inhibitors mediate suppression of alloreactive T cells and protection from GvHD by inducing CD8+ Treg. Because Cdk-mediated EZH2 phosphorylation is a key mechanism governing EZH2 function to regulate epigenetic silencing, Cdk2 inhibition might have additional, yet unidentified implications on gene expression programs of alloreactive T cells. Disclosures: No relevant conflicts of interest to declare.

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Apoptotic Cells for the Prevention of Cytokine Release Syndrome (CRS) in CAR T-Cell Therapy

Blood ◽

10.1182/blood.v128.22.1626.1626 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1626-1626

Author(s):

Dror Mevorach ◽

Veronique Amor ◽

Yehudith Shabat

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Inflammatory Cytokines ◽

Apoptotic Cells ◽

Car T ◽

Ifn Γ ◽

Pro Inflammatory Cytokines

Abstract Background: Chimeric antigen receptor (CAR)-modified T cells with specificity against CD19 have demonstrated dramatic promise against highly refractory hematologic malignancies. Clinical responses with complete remission rates as high as 90% have been reported in children and adults with relapsed/refractory acute lymphoblastic leukemia (ALL). However, very significant toxicity has been observed and as many as 30% in average developing severe forms of CRS and possibly related neurotoxicity. CRS is occurring due to large secretion of pro-inflammatory cytokines, mainly from macrophages/monocytes, and resembles macrophage-activating syndrome and hemophagocytosis in response to CAR T-secreting IFN-g and possibly additional cytokines. To better understand the mechanisms leading to CRS and to treat or prevent it, we have developed in vitro and in vivo models of CRS with and without CAR-modified T cells. Early apoptotic cells that have been successfully tested for the prevention of acute GVHD, including in 7 ALL patients, were tested in these models for their effect on cytokines and CAR T cell cytotoxicity. Methods: CD19-expressing HeLa cells were used alone or with co-incubation with human macrophages for in vitro experiments and intraperitoneal experiments. Raji was used in vivo for leukemia induction. LPS and IFN-γ were used to trigger additional cytokine release. CD19-specific CAR-modified cells were used (ProMab) for anti-tumor effect against CD19-bearing cells. Cytotoxicity assay was examined in vivo using 7-AAD with flow cytometry and in vitro by survival curves and analysis of tumor load in bone marrow and liver. CRS occurred spontaneously or in response to LPS and IFN-γ. Mouse IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15/IL-15R, and IL-7, as well as 32 human cytokines were evaluated by Luminex technology using the MAPIX system analyzer (Mereck Millipore) and MILLIPLEX Analyst software (Merek Millipore). Mouse IL-6Rα, MIG (CXCL9), and TGF-β1 were evaluated by Quantikine ELISA (R&D systems). Bone marrow and liver were evaluated using flow cytometry and immunohistochemistry. The IFN-γ effect was evaluated by STAT1 phosphorylation and biological products. Human macrophages and dendritic cells were generated from monocytes. Early apoptotic cells were produced as shown in GVHD clinical trial; at least 50% of cells were annexin V-positive and less than 5% were PI-positive. Results: Apoptotic cells had no negative effect in vitro or in vivo on CAR-modified T cells with specificity against CD19. There were comparable E/T ratios for CAR T in the presence or absence of apoptotic cells in vitro, and comparable survival curves in vivo. On the other hand, significant downregulation (p<0.01) of pro-inflammatory cytokines, including IL-6, IP-10, TNF-a, MIP-1α, MIP-1β, was documented. IFN-γ was not downregulated, but its effect on macrophages and dendritic cells was inhibited at the level of phosphorylated STAT1 and IFN-γ-induced expression of CXCL10 and CXCL9 was reduced. Conclusion: CRS evolves from several factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and as a response to the CAR T cell effect and expansion. Apoptotic cells decrease pro-inflammatory cytokines that originate from innate immunity and inhibit the IFN-γ effect on monocyte/macrophages/ dendritic cells without harming IFN-γ levels or CAR-T cytotoxicity. Disclosures Mevorach: Enlivex: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Amor:Enlivex: Employment. Shabat:Enlivex: Employment.

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Prostaglandin E2 directly inhibits the conversion of inducible regulatory T cells through EP2 and EP4 receptors via antagonizing TGF-β signalling

10.1101/2021.04.19.440391 ◽

2021 ◽

Author(s):

Marie Goepp ◽

Siobhan Crittenden ◽

You Zhou ◽

Adriano G Rossi ◽

Shuh Narumiya ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Prostaglandin E2 ◽

Th17 Cells ◽

Foxp3 Expression ◽

Treg Cells ◽

Itreg Cell

Background and Purpose: Regulatory T (Treg) cells are essential for control of inflammatory processes by suppressing Th1 and Th17 cells. The bioactive lipid mediator prostaglandin E2 (PGE2) promotes inflammatory Th1 and Th17 cells and exacerbates T cell-mediated autoimmune diseases. However, the actions of PGE2 on the development and function of Treg cells, particularly under inflammatory conditions, are debated. In this study, we examined whether PGE2 had a direct action on T cells to modulate de novo differentiation of Treg cells. Experimental Approach: We employed an in vitro T cell culture system of TGF-β-dependent Treg induction from naive T cells. PGE2 and selective agonists for its receptors, and other small molecular inhibitors were used. Mice with specific lack of EP4 receptors in T cells were used to assess Treg cell differentiation in vivo. Human peripheral blood T cells from healthy individuals were used to induce differentiation of inducible Treg cells. Key Results: TGF-β-induced Foxp3 expression and Treg cell differentiation in vitro was markedly inhibited by PGE2, which was due to interrupting TGF-β signalling. EP2 or EP4 agonism mimicked suppression of Foxp3 expression in WT T cells, but not in T cells deficient in EP2 or EP4, respectively. Moreover, deficiency of EP4 in T cells impaired iTreg cell differentiation in vivo. PGE2 also appeared to inhibit the conversion of human iTreg cells. Conclusion and Implications: Our results show a direct, negative regulation of iTreg cell differentiation by PGE2, highlighting the potential for selectively targeting the PGE2-EP2/EP4 pathway to control T cell-mediated inflammation.

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Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

Circulation Research ◽

10.1161/res.129.suppl_1.p482 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Anichavezhi Devendran ◽

Rasheed Bailey ◽

Sumanta Kar ◽

Francesca Stillitano ◽

Irene Turnbull ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Mononuclear Cells ◽

Embryonic Stem ◽

Model Systems ◽

Patient Specific ◽

Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

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Role of STAT3 in Immunoregulatory Function of Human Bone Marrow-Derived Mesenchymal Stem Cells.

Blood ◽

10.1182/blood.v114.22.3637.3637 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3637-3637

Author(s):

Jinsun Yoon ◽

Seoju Kim ◽

Eun Shil Kim ◽

Byoung Kook Kim ◽

Young Lee

Keyword(s):

T Cells ◽

Conflicts Of Interest ◽

Hematologic Malignancies ◽

Treg Cells ◽

Human Mscs ◽

Hematopoietic Stem ◽

The One

Abstract Abstract 3637 Poster Board III-573 The one of the best curative treatment modality in hematologic malignancies is an allogeneic hematopoietic stem cell transplantation (HSCT). However, graft-versus-host disease (GVHD) is a major obstacle of allogeneic HSCT. BM derived human MSCs are known to have immunoregulatory effect in vitro and in vivo via inhibiting alloreactive T lymphocytes, leading to their clinical use for the prevention of GVHD in HSCT. However, the molecular mechanism of immunoregulatory effect of human MSCs is not fully understood. In this study, the signaling of immunoregulatory effect was investigated by co-culture of human MSCs with lymphocytes. The proliferation of allogeneic T cells was inhibited by MSCs. Among the STATs, STAT3 was a key molecule in MLR co-cultured with MSCs. STAT3 siRNA treated MSCs did not inhibit the lymphocyte proliferation. After MSCs were trasnsfected with STAT3 plasmid, the fraction of CD4+CD25+FOXP3+ cells (Treg cells) were increased, while the fraction of CD4+, CD8+, CD25+ was decreased. In addition, Th1-related cytokines (IL-2, IL-12 and INF-γ) and Th17-related cytokines (IL-6, IL-17 and IL-21) were down-regulated, and Th2-related cytokines (GATA-3, IL-4 and IL-10) were up-regulated in MLR co-cultured with STAT3-ablated MSCs, while vice versa in MLR co-cultured with STAT3-transfected MSCs. Furthermore, ELISA showed that concentration of Th1-related cytokine (IL-2) in the supernatant of MLR co-cultured with STAT3-ablated MSCs was higher than that of control; while concentration of Th2-related cytokine (IL-4) was lower than that of control. These results suggested that induction of Th1 to Th2 shift by MSCs might be mediated via STAT3 molecule. In summary, STAT3 may be an indispensable molecule in the immunoregulatory effect in human MSCs via modulation of regulatory T cells. Disclosures: No relevant conflicts of interest to declare.

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In Vitro expanded STAT1−/− Regulatory T Cells Prevent Acute Graft Versus Host Disease (GVHD) and Promote Donor Cell Engraftment In Allogeneic Fully MHC-Mismatched Bone Marrow Transplant

Blood ◽

10.1182/blood.v116.21.732.732 ◽

2010 ◽

Vol 116 (21) ◽

pp. 732-732

Author(s):

Huihui Ma ◽

Caisheng Lu ◽

Judy Ziegler ◽

Suzanne Lentzsch ◽

Markus Y Mapara

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Cells ◽

Donor Cell ◽

Treg Cells ◽

Cell Engraftment ◽

Lethal Irradiation

Abstract Abstract 732 Treg cells have been recognized as critical regulators of the immune response and shown to prevent the development of GVHD. However, little is known about of the role of STAT1 signaling in Treg cells during the development of GVHD. In this study, we tried to investigate how STAT1 signaling controls donor Treg development and function in the setting of GVHD. For this purpose we studied the role of STAT1 in natural and inducible Treg (nTreg and iTreg, respectively). To better understand the influence of STAT1-deficiency on the proliferation of nTreg cells, purified splenic STAT1−/− or STAT1+/+ CD4+CD25+ cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and cultured on anti-CD3 coated plates in the presence of anti-CD28 and IL-2 for 3 days and analyzed for proliferation and viability. After 72h of in vitro culture 50% of the STAT1+/+ starting population were no longer viable compared to only 10% of STAT1−/− cells. Furthermore, we noted a significantly increased expansion of STAT1-deficient CD4+CD25+Foxp3+ Treg cells compared to STAT1+/+ Treg cells (p<0.001). In line with these findings, STAT1-deficiency resulted in a significantly higher proportion of CFSElo cells indicating vigorous proliferation (85% Foxp3+CFSElo in STAT1−/− compared to only 65% Foxp3+CFSElo in STAT1+/+ Treg cells. Furthermore, at the end of the culture 30% of the STAT1+/+ CD4+CD25+ population were Foxp3-negative compared to only 10% of the STAT1−/− cells. We next determined the impact of STAT1 on the generation of iTreg cells in vitro. For this purpose CD4+CD25− cells from STAT1−/− or STAT1+/+ mice were cultured for 3 days on anti-CD3 coated plates in the presence of anti-CD28 antibodies, hTGF-β, mIL-2, anti-IFN-γ and anti-IL-4 for 3 days. Compared to STAT1+/+, we observed significantly enhanced generation of iTregs from STAT1−/− splenocytes (19.9%±3.0% vs. 10.6%±1.3%, p=0.008). We then performed studies to assess the in vivo generation of iTreg. For that purpose BALB/c mice were reconstituted with T Cell Depleted (TCD) 129.STAT1+/+Bone Marrow Cells (BMC) following lethal irradiation and recipients were co-injected with CD4+CD25− cells purified from either 129.STAT1+/+ or 129.STAT1−/− splenocytes. We again noted a significantly higher proportion of CD4+CD25+ Foxp3+ cells in recipients of CD4+CD25−STAT1−/− cells compared to recipients of STAT1+/+ T cells indicating a significantly increased conversion of CD4+CD25- cells into Treg cells. To confirm the in vitro results we tested the functional ability of in vitro expanded (using anti-CD3, anti-CD28, IL-2 and TGF-β) STAT1+/+ or STAT1−/− Treg cells to block induction of GVHD. GVHD was induced in BALB/c mice following lethal irradiation (800rad) and fully MHC-mismatched BMT using 129.STAT1+/+ bone marrow cells plus 129.STAT+/+ conventional T cells (Tcon). Animals were co-injected with expanded Treg cells from either 129.STAT1+/+ or 129.STAT1−/− donors at a ratio of 1:1 or 1:4 (Treg:Tcon). STAT1−/− or STAT1+/+ Treg cells were equipotent in completely preventing GVHD mortality. However, compared to recipients of STAT1+/+ Treg recipients of STAT1−/− Treg showed reduced signs of GVHD morbidity as determined by a significantly improved weight development. Furthermore, recipients of STAT1−/− Treg showed significantly increased donor cell engraftment compared to recipients of STAT1+/+Treg (donor CD4+ [87% vs. 60%, p=0.03], CD8+[99% vs. 96%, p=0.04], Mac1+[96% vs. 77%, p=0.02] and B220+[100% vs. 96%, p=0.007]) cells in the recipient spleen. These observations clearly demonstrate that STAT1 is a critical regulator of Treg cell development and expansion and that targeting STAT1 in CD4+ T cells may facilitate in vitro and in vivo generation/expansion of Treg cells for therapeutic use in GVHD while also promoting donor cell engraftment. Disclosures: Lentzsch: Celgene Corp: Research Funding. Mapara:Resolvyx: Research Funding; Gentium: stocks.

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Lenalidomide Redresses the Effector T Cell:Treg Cell Imbalance in Patients with Multiple Myeloma.

Blood ◽

10.1182/blood.v120.21.2977.2977 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2977-2977

Author(s):

Clive R Carter ◽

Christopher Parrish ◽

Sylvia Feyler ◽

Natuley Smalle ◽

Gina B Scott ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Flow Cytometric Analysis ◽

Treg Cells ◽

Hematological Toxicity ◽

Complete Medium ◽

Circulating Lymphocytes ◽

Pre Treatment

Abstract Abstract 2977 Rationale. Competent immune responses require balanced effector and regulatory arms; cancer may influence either in an attempt to evade immune control. Pharmaco-immunotherapy seeks to redress this imbalance. The mode of action of lenalidomide (Len) in multiple myeloma (MM) is poorly characterized, but is often affirmed to comprise direct antitumor and immunostimulatory effects. Using a combination of in vivo and in vitro methods we analyzed the effect of Len and dexamethasone (Dex) on immune reconstitution in MM patients. Methods. 21 patients with relapsed MM were treated with LenDex, commencing at Len 25mg/day PO D1–21 and Dex either 480mg (n=5) or 160mg (n=16) PO per 28 day cycle. Dex doses were tapered after cycle 6; Len doses were adjusted for renal function and hematological toxicity. Peripheral blood (PB) samples were analyzed at baseline and after cycles 1, 4, 7 and 10. PB from age-matched healthy volunteers (n=14) was also analyzed. For in vitro work, the human MM cell line (HMCL) U266B was cultured with naive CD4+CD25− T cells at a 1:2 ratio in complete medium (no additional cytokines). Where indicated, U266B was incubated with Len for 24 hours, then washed prior to coculture. Cells were harvested after 7 days for flow cytometric analysis. Results. Coculture of HMCL with sorted CD4+CD25− T cells in vitro induced Treg (CD4+CD25BrightFoxP3+) differentiation (31.6% vs. 0.7% without HMCL, n=10, p<0.001), with only minimal effector T cell (Teff) generation (CD4+CD25+FoxP3−). Incubation of HMCL with Len prior to coculture selectively suppressed Treg cell generation (Len: 3.2±1.1%, DMSO (vehicle): 36±4.4%, p<0.001), giving rise to a higher Teff:Treg ratio. Apoptosis in HMCL after coculture was similar in Len- and DMSO-treated cells (Len: 38.2±0.9%, DMSO: 39.35±0.8%, p=NS). Patients with MM showed profound CD4+ lymphopenia (MM: 369±82 cells/μl, controls: 700±182 cells/μl; p=0.027), which was not corrected by LenDex, despite steroid tapering. The number of recent thymic emigrants (RTE: CD4+CD45RA+CD31+) in MM patients was significantly lower than in control subjects (MM: 37.4±9.9/μl, control: 159±25.1/μl, p=0.0002). A significant reduction in the proportion of RTE was seen after cycle 1 of LenDex (11+2.2%, p=0.028) with evidence of recovery toward normal both in terms of proportion (17.4+3.4%, p=0.48) and absolute numbers (65.13+3.5, p=0.042) by cycle 7. We examined the activation status of circulating lymphocytes and noted a significant increase in the proportion of CD3+ cells expressing HLA-DR compared with controls (50.6+4% vs. 17.7+2.8%, p<0.0001); this was unaffected by LenDex. In contrast, activated CD4+ Teff cells (CD4+CD25+FoxP3−) were significantly reduced in the PB of MM patients pre-treatment compared to controls (26.1+5.1% vs. 45.6+2.3%, p<0.001). LenDex increased the proportion of activated CD4+ T cells (1-way ANOVA p=0.021); this was predominantly seen between cycles 4 and 7. As has been reported previously, Treg cells as a proportion of CD4+ cells were expanded in MM patients (MM: 10.92±2.7%, controls: 3.24±0.3%, p=0.005), although absolute counts were not significantly lower (MM: 45.5±15/μl, controls: 67.3±8/μl, p=0.181). Treg cells rapidly fell early in LenDex therapy, but returned to baseline in association with steroid tapering. There was a non-significant correlation between the baseline level of Treg cells and time to progression (TTP; r2=0.707, p=0.08) and a significant correlation between the level of Treg cells after cycle 4 and TTP (r2=0.669, p=0.009). The Teff:Treg ratio in patients at baseline was lower than in controls (9.2+3 vs 14.8+1, p=0.05). Whilst an early reduction in the ratio was seen with higher steroid dosing, the ratio rose above baseline by cycle 4 (9.2+3 vs. 11.4+3.6; p=NS). Neither baseline (p=0.29) nor cycle 4 (p=0.22) ratios correlated with TTP. Conclusions. Patients with MM exhibit marked immune dysfunction, particularly characterized by imbalance between the effector and regulatory arms of the immune response. Using an in vitro culture system we show lenalidomide abrogates the ability of MM cells to drive Teff:Treg imbalance. Furthermore, in the clinic, LenDex therapy for patients with relapsed MM leads to correction of the Teff:Treg ratio. Further appraisal is now required to allow correlation of this immunotherapeutic effect with clinical outcomes. Disclosures: Cook: Celgene: Consultancy; Janssen: Consultancy.

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