Fli-1 Regulates Multiple T-Cell Subsets during Inflammatory Responses and Experimental Graft-Versus-Host Disease

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a procedure undertaken to cure hematological malignancies, especially leukemia. Graft-Versus-Host Disease (GVHD) is a serious condition that often appears following allo-HCT. Friend Leukemia Virus Integration 1 (Fli-1) is a transcription factor highly expressed in cancers including erythroleukemia and acute myeloid leukemia while also implicated in pathogenesis of systemic lupus. We have interrogated the role of Fli-1 in T-cell responses by generating a novel T-cell specific conditional disruption of fli-1 where essential exons 3 and 4 of the gene are floxed and excised in the presence of CD4-Cre recombinase. Models of acute GVHD (aGVHD) and chronic GVHD (cGVHD) were tested utilizing hematopoietic cells from mice with a heterozygous (fli-1-/+ or Het) or homozygous (fli-1-/-or Null) disruption of the fli-1 gene, or from wild-type (fli-1+/+ or WT) littermate controls. At baseline, T cells among each of these three mouse strains showed no significant difference in CD44/CD62L expression or CD4+FoxP3+ (nTreg) frequencies. In the cGVHD model, BALB/c (H2Kd) recipients were infused with allogeneic B6 (H2Kb) genotype-respective bone marrow and splenocytes in order to induce cGVHD. Recipients that received fli-1-/+CD4Cre+ marrow and splenocytes demonstrated improved survival and mild cGVHD, whereas those that received fli-1-/-CD4Cre+ or WT donor cells developed serve cGVHD (Fig. 1 a-f). Cellular studies from lymphoid organs of cGVHD allo-HCT recipients revealed that disruption of fli-1 was associated with decreased frequencies of donor CD4+ cells expressing IL-17A, IFN-γ, T follicular-like (TFH-like) cell markers, and CD8+ cells expressing PD-1. In aGVHD settings, donor fli-1-/+CD4Cre+ T cells had a decreased ability to induce aGVHD compared to WT donor T cells and fli-1-/- donor T cells (Fig. 2 a-b). When investigating cellular mechanisms in aGVHD settings, we found that fli-1-/+CD4Cre+ T cells produced significantly lower IFN-γ early after T-cell activation in vivo compared to WT and fli-1-/-CD4Cre+ T cells (Fig. 2 c). To then investigate the role of Fli-1 in T cells beyond GVHD, we utilized a colitis model by transferring naïve CD4+ T cells (CD44-CD25-) into Rag2-/- syngeneic recipients. While fli-1+/+ T cells induced severe colitis as expected, fli-1-/- and fli--/+ T cells caused moderate and mild colitis, respectively. These results were consistent with those observed in GVHD models. To elucidate underlying mechanisms, we tested the effects of Fli-1 on antigen-specific T-cells using a MHC-II restricted TCR transgenic (Tg) mouse strain specific for HY-peptide. CD4+CD25- T cells from fli-1+/+, fli-1-/+, or fli-1-/- CD4Cre+ TCR-Tg mice were polarized in vitro with endogenous antigen presenting cells from spleen in presence of HY-peptide under iTreg- or Th17-polarizing conditions. Both fli-1-/+ and fli-1-/- TCR-tg T cells exhibited significant increases in iTreg (CD4+FoxP3+) frequencies and surface expression of iTreg functional markers (CD25, CD39, CD73, NRP-1), while also having significantly decreased frequencies of IL-17A producing T cells compared to WT-TCR-tg T cells. To further explore the molecular mechanisms, we retrieved fli-1+/+, fli-1-/+, or fli-1-/- donor T cells from recipient spleens after allo-HCT and did RNAseq analysis on these cells. RNAseq data reveals significant differences in mRNA expression within acute inflammatory response and positive regulation of the immune response enrichment pathways between fli-1-/+ T cells and littermate fli-1+/+ T cells, and to a lesser extent in fli-1-/- T cells (Fig. 2 d-f). Thus, reducing Fli-1 transcriptional activity represents a potential therapeutic concept toward ameliorating GVHD after allo-HCT, while simultaneously targeting cancers such as leukemia which typically overexpress Fli-1. Disclosures No relevant conflicts of interest to declare.

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Interleukin-18 Regulates Acute Graft-Versus-Host Disease by Enhancing Fas-mediated Donor T Cell Apoptosis

Journal of Experimental Medicine ◽

10.1084/jem.194.10.1433 ◽

2001 ◽

Vol 194 (10) ◽

pp. 1433-1440 ◽

Cited By ~ 132

Author(s):

Pavan Reddy ◽

Takanori Teshima ◽

Mark Kukuruga ◽

Rainer Ordemann ◽

Chen Liu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Acute Gvhd ◽

Host Disease ◽

Murine Bone Marrow ◽

Graft Versus Host ◽

Donor T Cells ◽

Ifn Γ ◽

Acute Graft

Interleukin (IL)-18 is a recently discovered cytokine that modulates both T helper type 1 (Th1) and Th2 responses. IL-18 is elevated during acute graft-versus-host disease (GVHD). We investigated the role of IL-18 in this disorder using a well characterized murine bone marrow transplantation (BMT) model (B6 → B6D2F1). Surprisingly, blockade of IL-18 accelerated acute GVHD-related mortality. In contrast, administration of IL-18 reduced serum tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS) levels, decreased intestinal histopathology, and resulted in significantly improved survival (75 vs. 15%, P < 0.001). Administration of IL-18 attenuated early donor T cell expansion and was associated with increased Fas expression and greater apoptosis of donor T cells. The administration of IL-18 no longer protected BMT recipients from GVHD when Fas deficient (lpr) mice were used as donors. IL-18 also lost its ability to protect against acute GVHD when interferon (IFN)-γ knockout mice were used as donors. Together, these results demonstrate that IL-18 regulates acute GVHD by inducing enhanced Fas-mediated apoptosis of donor T cells early after BMT, and donor IFN-γ is critical for this protective effect.

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Bone Marrow NK1.1− and NK1.1+ T Cells Reciprocally Regulate Acute Graft versus Host Disease

Journal of Experimental Medicine ◽

10.1084/jem.189.7.1073 ◽

1999 ◽

Vol 189 (7) ◽

pp. 1073-1081 ◽

Cited By ~ 239

Author(s):

Defu Zeng ◽

David Lewis ◽

Sussan Dejbakhsh-Jones ◽

Fengshuo Lan ◽

Marcos García-Ojeda ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Cd8 T Cells ◽

Peripheral Blood ◽

T Cell Subsets ◽

Host Disease ◽

Graft Versus Host ◽

Ifn Γ

Sorted CD4+ and CD8+ T cells from the peripheral blood or bone marrow of donor C57BL/6 (H-2b) mice were tested for their capacity to induce graft-versus-host disease (GVHD) by injecting the cells, along with stringently T cell–depleted donor marrow cells, into lethally irradiated BALB/c (H-2d) host mice. The peripheral blood T cells were at least 30 times more potent than the marrow T cells in inducing lethal GVHD. As NK1.1+ T cells represented <1% of all T cells in the blood and ∼30% of T cells in the marrow, the capacity of sorted marrow NK1.1− CD4+ and CD8+ T cells to induce GVHD was tested. The latter cells had markedly increased potency, and adding back marrow NK1.1+ T cells suppressed GVHD. The marrow NK1.1+ T cells secreted high levels of both interferon γ (IFN-γ) and interleukin 4 (IL-4), and the NK1.1− T cells secreted high levels of IFN-γ with little IL-4. Marrow NK1.1+ T cells obtained from IL-4−/− rather than wild-type C57BL/6 donors not only failed to prevent GVHD but actually increased its severity. Together, these results demonstrate that GVHD is reciprocally regulated by the NK1.1− and NK1.1+ T cell subsets via their differential production of cytokines.

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Dynamic regulation of effector IFN-γ-producing and IL-17-producing T cell subsets in the development of acute graft-versus-host disease

Molecular Medicine Reports ◽

10.3892/mmr.2015.4638 ◽

2015 ◽

Vol 13 (2) ◽

pp. 1395-1403 ◽

Cited By ~ 8

Author(s):

KAI ZHAO ◽

SUHONG RUAN ◽

LINGLING YIN ◽

DONGMEI ZHAO ◽

CHONG CHEN ◽

...

Keyword(s):

T Cell ◽

Graft Versus Host Disease ◽

T Cell Subsets ◽

Dynamic Regulation ◽

Host Disease ◽

Graft Versus Host ◽

Ifn Γ ◽

Acute Graft

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Critical role of host γδ T cells in experimental acute graft-versus-host disease

Blood ◽

10.1182/blood-2004-10-4087 ◽

2005 ◽

Vol 106 (2) ◽

pp. 749-755 ◽

Cited By ~ 37

Author(s):

Yoshinobu Maeda ◽

Pavan Reddy ◽

Kathleen P. Lowler ◽

Chen Liu ◽

Dennis Keith Bishop ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Critical Role ◽

Γδ T Cells ◽

Allogeneic Bone ◽

Host Disease ◽

Graft Versus Host ◽

Tnf Α

Abstract γδ T cells localize to target tissues of graft-versus-host disease (GVHD) and therefore we investigated the role of host γδ T cells in the pathogenesis of acute GVHD in several well-characterized allogeneic bone marrow transplantation (BMT) models. Depletion of host γδ T cells in wild-type (wt) B6 recipients by administration of anti-T-cell receptor (TCR) γδ monoclonal antibody reduced GVHD, and γδ T-cell-deficient (γδ-/-) BM transplant recipients experienced markedly improved survival compared with normal controls (63% vs 10%, P < .001). γδ T cells were responsible for this difference because reconstitution of γδ-/- recipients with γδ T cells restored GVHD mortality. γδ-/- recipients showed decreased serum levels of tumor necrosis factor α (TNF-α), less GVHD histopathologic damage, and reduced donor T-cell expansion. Mechanistic analysis of this phenomenon demonstrated that dendritic cells (DCs) from γδ-/- recipients exhibited less allostimulatory capacity compared to wt DCs after irradiation. Normal DCs derived from BM caused greater allogeneic T-cell proliferation when cocultured with γδ T cells than DCs cocultured with medium alone. This enhancement did not depend on interferon γ (IFN-γ), TNF-α, or CD40 ligand but did depend on cell-to-cell contact. These data demonstrated that the host γδ T cells exacerbate GVHD by enhancing the allostimulatory capacity of host antigen-presenting cells. (Blood. 2005;106:749-755)

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Variable capacity of L3T4+ T cells to cause lethal graft-versus-host disease across minor histocompatibility barriers in mice.

Journal of Experimental Medicine ◽

10.1084/jem.165.6.1552 ◽

1987 ◽

Vol 165 (6) ◽

pp. 1552-1564 ◽

Cited By ~ 120

Author(s):

R Korngold ◽

J Sprent

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Histocompatibility Antigen ◽

T Cell Subsets ◽

Minor Histocompatibility Antigen ◽

Host Disease ◽

Graft Versus Host ◽

Variable Capacity ◽

High Incidence

Highly purified populations of L3T4+ and Lyt-2+ T cell subsets were compared for their capacity to cause lethal GVHD in six different H-2-compatible, multiple minor histocompatibility antigen-different murine strain combinations. In four of these combinations (C3H.SW----B6, DBA/2----B10.D2, B10.BR----CBA, and B10.S----SJL), lethal GVHD appeared to be caused almost entirely by Lyt-2+ cells; the injection of L3T4+ cells resulted in low mortality even when these cells were presensitized to the recipient antigens. In the remaining two combinations (B10.D2----DBA/2 and B10.D2----BALB/c), L3T4+ T cells were able to cause a high incidence of GVHD and were more potent than the Lyt-2+ cells. The implications of these findings are discussed.

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Dendritic cell–activated CD44hiCD8+ T cells are defective in mediating acute graft-versus-host disease but retain graft-versus-leukemia activity

Blood ◽

10.1182/blood-2003-09-3135 ◽

2004 ◽

Vol 103 (10) ◽

pp. 3970-3978 ◽

Cited By ~ 76

Author(s):

Yi Zhang ◽

Gerard Joe ◽

Jiang Zhu ◽

Richard Carroll ◽

Bruce Levine ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dendritic Cell ◽

Graft Versus Host Disease ◽

T Cell Subsets ◽

Effector Memory ◽

Antitumor Effects ◽

Host Disease ◽

Graft Versus Host

Abstract Graft versus host disease (GVHD) is triggered by host antigen-presenting cells (APCs) that activate donor T cells to proliferate and differentiate, but which APC-activated donor T-cell subsets mediate GVHD versus beneficial antitumor effects is not known. Using a CD8+ T cell–dependent mouse model of human GVHD, we found that host dendritic cell (DC)–induced CD44hiCD8+ effector/memory T cells were functionally defective in inducing GVHD, whereas CD44loCD8+ naive phenotype T cells were extremely potent GVHD inducers. Depletion of CD44loCD8+ T cells from host DC-stimulated T cells before transplantation prevented GVHD without impairing their antitumor activity in vivo. Compared with CD44loCD8+ T cells, CD44hiCD8+ T cells expressed high levels of Fas and were efficiently deleted in vivo following transplantation. These results suggest that ex vivo allogeneic DC stimulation of donor CD8+ T cells may be useful for the prevention of GVHD and for optimizing antitumor therapies in vivo.

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β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

Blood ◽

10.1182/blood-2019-123139 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1915-1915 ◽

Cited By ~ 1

Author(s):

Hemn Mohammadpour ◽

Joseph L. Sarow ◽

George L. Chen ◽

Cameron R. MacDonald ◽

Umesh Sharma ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Adrenergic Receptor ◽

Graft Versus Host ◽

Graft Versus Leukemia ◽

Donor T Cells ◽

Β2 Adrenergic Receptor ◽

Ifn Γ

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P< 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P<0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No relevant conflicts of interest to declare.

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Patient Bone Marrow Chimerism at Time of Donor Lymphocyte Infusion Predicts Development of Graft Versus Host Disease

Blood ◽

10.1182/blood-2018-99-113839 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2132-2132

Author(s):

Eva AS Koster ◽

Liesbeth C. de Wreede ◽

Sylwia Wallet-Malicka ◽

Lisette Bogers ◽

Peter van Balen ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Hazard Ratio ◽

The State ◽

Host Disease ◽

Graft Versus Host ◽

Donor T Cells ◽

Donor Type

Abstract After allogeneic stem cell transplantation (alloSCT), donor T cells targeting patient derived hematopoietic cells can induce a Graft versus Leukemia (GvL) effect preventing relapse. However, targeting of healthy patient tissues can cause Graft versus Host Disease (GvHD). The inflammatory environment induced by pre transplantation conditioning, the number of donor T cells in the graft, genetic disparity between patient and donor and the presentation of allo-antigens by activated patient derived antigen-presenting cells (APC) to donor T cells play a role in the development of GvL and/or GvHD. Donor T cell depletion (TCD) reduces GvHD and GvL. After TCD alloSCT, postponed prophylactic donor lymphocyte infusions (pDLI) are often needed to induce a GvL effect. When using 10/10 matched donors, our first dose of pDLI at six months after TCD alloSCT contained 3.0x10^6 T cells/kg (related donor, RD) or 1.5x10^6 T cells/kg (unrelated donor, UD). We evaluated whether the risk of developing GvHD after DLI is influenced by the donor type, intensity of the conditioning and/or patient bone marrow (BM) chimerism at time of DLI Sixty patients with acute leukemia (52 AML, 8 ALL; median age 57; 27 RD, 33 UD) received pDLI at a median of 6.4 months after TCD alloSCT in the absence of GVHD or relapse. Twenty-four patients received myeloablative (MA) conditioning consisting of cyclophosphamide and TBI. 36 patients received non-myeloablative (NMA) conditioning based on fludarabin and busulphan. TCD was performed by adding 20mg alemtuzumab to the graft. Only MA conditioned patients with an UD (n=12) received post transplantation ciclosporin as GvHD prophylaxis, which was tapered from 1 month after alloSCT. Clinically significant GvHD was defined as need of therapeutic systemic immunosuppression (tIS) for GvHD for at least 2 weeks or until death. Bone Marrow (BM) chimerism was measured prior to DLI. Three categories of patient chimerism levels were defined: no patient derived cells (absent), patient derived cells present, but < 5% (low), or ≥ 5% (high). In case of persisting or increasing patient chimerim after pDLI, a second DLI was given at 3-6 months after the first. A multi-state model was designed (Figure 1) with the first DLI (DLI1) as starting state and time. Patients starting tIS after DLI1 transit to the state tIS. Patients who need a second DLI, develop a relapse or die, transit to these respective states. Patients who stay in the state of DLI1 are considered to have a positive outcome. All patients had a follow-up of at least one year after DLI. Numbers in the boxes in Figure 1 represent the number of patients in that state at 1 year after DLI1 and numbers next to the arrows indicate the numbers of patients who made the transition between the two states. Donor type (unrelated versus related), conditioning (NMA versus MA) and patient BM chimerism at time of DLI were included in a Cox model for the transition hazards to investigate their association with the development of GvHD after DLI. For the total group, the cumulative incidence of tIS at 1 year after pDLI was 33% (95% CI 21-45%). Patients with an UD had a hazard ratio (HR) of 1.1 (95% CI 0.4-3.3) of needing tIS after DLI1 compared to patients with a RD. Compared to MA conditioning, NMA conditioned patients had a hazard ratio of 2.1 (95% CI 0.5-8.9) of needing tIS after DLI. They had a HR of 0.2 (95% CI 0.04-0.95) of stopping tIS compared to MA conditioned patients, indicating that DLI after NMA conditioning is associated with more severe GVHD. We hypothesized that this was due to the persistence of patient derived APC. BM chimerism at time of DLI was measured in 47 patients. After NMA and MA conditioning, BM patient chimerism was absent in 14% and 56%, low in 41% and 39%, and high in 45% and 6%, respectively (Fisher's exact test p=0.002 for difference between type of conditioning). Compared to the group without patient chimerism, the low and high patient chimerism group had a HR of 1.9 (95% CI 0.9-4.2) and 3.6 (95% CI 1.7-8.0) of needing tIS after DLI, respectively (Figure 2), demonstrating that the level of patient chimerism is a strong predictor for development of GvHD after DLI, even when taking into account the type of conditioning regimen. Patient BM chimerism at time of pDLI is a strong and independent predictor for the risk of developing GvHD. Dose reduction in case of an UD equalized the GvHD risk compared to a RD. When choosing a T cell dose for pDLI, patient chimerism should be considered a relevant parameter. Disclosures No relevant conflicts of interest to declare.

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Inhibition of Graft-Versus-Host Disease (GVHD) by Histone Deacetylase Inhibitor (HDAC) SAHA Leads to Downregulation of STAT1 Activation.

Blood ◽

10.1182/blood.v106.11.1311.1311 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1311-1311

Author(s):

Corinna Leng ◽

Cuiling Li ◽

Judy Ziegler ◽

Anna Lokshin ◽

Suzanne Lentzsch ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Histone Deacetylase ◽

Graft Versus Host Disease ◽

Host Disease ◽

Graft Versus Host ◽

Tnf Α ◽

Ifn Γ

Abstract Histone deacetylase (HDAC) inhibitors have been shown to reduce development of graft versus host disease [GVHD] following allogeneic bone marrow transplantation [BMT]. Administration of the HDAC inhibitor suberonylanilide hydroxamic acid [SAHA] resulted in a significantly reduced GVHD-dependent mortality following fully MHC-mismatched allogeneic BMT. Median Survival Time (MST) for vehicle and SAHA-treated mice were 7.5 days and 38 days respectively. However, SAHA treatment did not affect T cell activation nor T cell expansion in vitro and in vivo as determined by MLR assays, phenotypic analysis of donor T cells with regard to expression of the CD25 activation antigen and calculation of donor CD4+ and CD8+ T cell numbers on days +3 and +6 post-BMT. Thus, SAHA treatment was not able to inhibit the strong upregulation of CD25 antigen on CD8+ T cells observed during induction of GVHD on days +3 and +6 post-BMT. We therefore focused on the effects of SAHA treatment on efferent immune effects including cytokine secretion and intracellular signaling events in vitro and in vivo following GVHD induction. SAHA treatment broadly inhibited lipopolysaccharide [LPS] and allo-antigen-induced cytokine/chemokine secretion in vitro like MIP-1-α, IP-10, IFN-γ, TNF-α and IL-6 and led also to a significant decrease in IFN-γ and TNF-α levels in vivo following induction of GVHD. Concomitantly, SAHA treatment inhibited phosphorylation of STAT1 and STAT3 in response to LPS and allo-activation in vitro. Furthermore, analysis of liver tissue and spleens from SAHA-treated animals with GVHD showed a significant decrease in phosphorylated STAT1. In contrast SAHA treatment had only moderate effects on p38 or ERK1,2 Mitogen-activated Protein Kinase (MAPK) pathway underscoring the relevance of the inhibition of the STAT1 pathway. In conclusion, GVHD is associated with a strong induction of phosphorylation of STAT1 in the liver and spleen and SAHA-dependent reduction of GVHD is associated with systemic and local inhibition of pSTAT1 and modulation of the inflammatory cytokine milieu during the efferent immune response.

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MiR-146a Regulates the TRAF6/TNF-Axis in Donor T Cells during Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v124.21.843.843 ◽

2014 ◽

Vol 124 (21) ◽

pp. 843-843

Author(s):

Natalie Stickel ◽

Gabriele Prinz ◽

Dietmar Pfeifer ◽

Annette Schmitt-Graeff ◽

Marie Follo ◽

...

Keyword(s):

T Cells ◽

Graft Versus Host Disease ◽

Acute Gvhd ◽

Negative Regulator ◽

Snp Analysis ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host ◽

Donor T Cells

Abstract Introduction: Acute graft-versus-host disease (GvHD) arises from the attack of recipient tissues by donor allogeneic T cells and represents one of the major limitations of allogeneic hematopoietic cell transplantation (allo-HCT). In spite of many clinical trials, the standard immunosuppressive regimens for prevention of acute GvHD have improved little in the last two decades. Hence, a better understanding of the biology of acute GvHD may improve therapeutic options. MicroRNA-146a (miR-146a) was found to be increased in the sera of patients with GvHD. Therefore, we aimed to decipher the role of miR-146a in allogeneic donor T cells during GvHD by functional studies and in patients undergoing allo-HCT by single nucleotide polymorphism (SNP) analysis. Methods: We used two different murine major MHC mismatch models for acute GvHD. Recipient mice were conditioned with irradiation before transplantation of bone marrow and either wildtype or miR-146a deficient T cells from allogeneic donor mice. Furthermore, genomic DNA from 289 patients that underwent allo-HCT and their respective hematopoietic stem cell donors was isolated in order to determine their miR-146a rs2910164genotype. Results: We observed miR-146a upregulation in T cells of mice developing acute GvHD compared to untreated mice in a major MHC and a minor histocompatibility antigen mismatch model. Transfer of miR-146a deficient T cells caused increased GvHD severity, elevated TNF serum levels and reduced survival. Conversely, the phytochemical induction of miR-146a or its overexpression in donor T cells using a specific miR-146a mimic reduced GvHD severity. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was upregulated in miR-146a-/- T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased NF-κB activity and TNF production in miR-146a-/- T cells, while other pro-inflammatory cytokine levels were unaffected. The detrimental effect of miR-146a deficiency in T cells could be antagonized by TNF blockade in vivo. Moreover, in contrast to WT T cells, over expression of miR-146a in Tnf deficient T cells had no effect on their alloreactivity. In the human system, the minor genotype of the SNP rs2910164, which causes reduced miR-146a expression, was more frequent in patients developing acute GvHD grade III/IV compared to all other allo-HCT recipients (n=289). Conclusions: Taken together we show that miR-146a functions as a negative regulator of the TRAF6/TNF-axis in allogeneic donor T cells during GvHD, leading to reduced TNF transcription. Given our observation on the predictive role of the SNP leading to decreased miR-146a expression in acute GvHD in patients and the possibility to exogenously enhance miR-146a expression, we provide a novel and targeted molecular approach to mitigate GvHD. Disclosures No relevant conflicts of interest to declare.

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