Antilymphocytic antibodies and marrow transplantation. XII. Suppression of graft-versus-host disease by T-cell-modulating and depleting antimouse CD3 antibody is most effective when preinjected in the marrow recipient

Abstract A hamster antimouse CD3 monoclonal antibody (MoAb) opened the way to experimental studies on the suppression of allograft rejection and cytokine-related morbidity after treatment with antibodies modulating the CD3/T-cell receptor complex (CD3/TCR). Because earlier attempts to suppress graft-versus-host disease (GVHD) in patients by in vitro treatment of donor marrow with anti-CD3 MoAb had remained inconclusive, we used a rat IgG2b antimouse CD3 MoAb (17A2) with fewer side effects to analyze suppression of GVHD in the mouse model. Detailed phenotyping of blood, spleen, and lymphnode T cells after the injection of 400 micrograms 17A2 in C57BL/6 mice showed 60% CD3 downmodulation and 50% T- cell depletion for spleen cells. Injection of these spleen cells, together with bone marrow cells, in fully mismatched preirradiated CBA mice delayed GVHD by only 6 days. Ex vivo treatment of donor cells with 17A2 was not effective. In contrast, conditioning of marrow recipients with a single injection of 17A2 delayed 50% GVHD mortality by 100 days and prevented GVHD altogether after prolonged treatment, with survivors showing complete chimerism and specific transplantation tolerance. This difference in antibody effect contrasts with earlier experiences with nonmodulating but more strongly T-cell-depleting MoAbs of the same isotype, which prevent GVHD no matter whether applied in vitro or injected into donor or recipient mice. Our data indicate that CD3/TCR reexpression in marrow recipients with no circulating 17A2 is the reason why ex vivo donor cell treatment with anti-CD3 MoAb is comparatively ineffective. Our data, which allow separate evaluation of cell-depleting and cell-modulating antibody activity, help to explain previous clinical failure to suppress GVHD and provide evidence in favor of conditioning the marrow recipient with anti-CD3 MoAb as a therapeutic alternative.

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Antilymphocytic antibodies and marrow transplantation. XII. Suppression of graft-versus-host disease by T-cell-modulating and depleting antimouse CD3 antibody is most effective when preinjected in the marrow recipient

Blood ◽

10.1182/blood.v80.10.2661.bloodjournal80102661 ◽

1992 ◽

Vol 80 (10) ◽

pp. 2661-2667 ◽

Cited By ~ 7

Author(s):

J Mysliwietz ◽

S Thierfelder

Keyword(s):

T Cell ◽

Graft Versus Host Disease ◽

Ex Vivo ◽

Experimental Studies ◽

Prolonged Treatment ◽

Antibody Activity ◽

Spleen Cells ◽

Host Disease ◽

Graft Versus Host

A hamster antimouse CD3 monoclonal antibody (MoAb) opened the way to experimental studies on the suppression of allograft rejection and cytokine-related morbidity after treatment with antibodies modulating the CD3/T-cell receptor complex (CD3/TCR). Because earlier attempts to suppress graft-versus-host disease (GVHD) in patients by in vitro treatment of donor marrow with anti-CD3 MoAb had remained inconclusive, we used a rat IgG2b antimouse CD3 MoAb (17A2) with fewer side effects to analyze suppression of GVHD in the mouse model. Detailed phenotyping of blood, spleen, and lymphnode T cells after the injection of 400 micrograms 17A2 in C57BL/6 mice showed 60% CD3 downmodulation and 50% T- cell depletion for spleen cells. Injection of these spleen cells, together with bone marrow cells, in fully mismatched preirradiated CBA mice delayed GVHD by only 6 days. Ex vivo treatment of donor cells with 17A2 was not effective. In contrast, conditioning of marrow recipients with a single injection of 17A2 delayed 50% GVHD mortality by 100 days and prevented GVHD altogether after prolonged treatment, with survivors showing complete chimerism and specific transplantation tolerance. This difference in antibody effect contrasts with earlier experiences with nonmodulating but more strongly T-cell-depleting MoAbs of the same isotype, which prevent GVHD no matter whether applied in vitro or injected into donor or recipient mice. Our data indicate that CD3/TCR reexpression in marrow recipients with no circulating 17A2 is the reason why ex vivo donor cell treatment with anti-CD3 MoAb is comparatively ineffective. Our data, which allow separate evaluation of cell-depleting and cell-modulating antibody activity, help to explain previous clinical failure to suppress GVHD and provide evidence in favor of conditioning the marrow recipient with anti-CD3 MoAb as a therapeutic alternative.

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A combination of anti-CD3 and anti-CD7 ricin A-immunotoxins for the in vivo treatment of acute graft versus host disease

Blood ◽

10.1182/blood.v95.12.3693 ◽

2000 ◽

Vol 95 (12) ◽

pp. 3693-3701 ◽

Cited By ~ 26

Author(s):

Ypke V. J. M. van Oosterhout ◽

Liesbeth van Emst ◽

Anton V. M. B. Schattenberg ◽

Wil J. M. Tax ◽

Dirk J. Ruiter ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Graft Versus Host Disease ◽

T Cell Response ◽

Cytotoxic T Cell ◽

Host Disease ◽

Graft Versus Host ◽

Ricin A

Abstract This study evaluated the anti-graft versus host disease (GVHD) potential of a combination of immunotoxins (IT), consisting of a murine CD3 (SPV-T3a) and CD7 (WT1) monoclonal antibody both conjugated to deglycosylated ricin A. In vitro efficacy data demonstrated that these IT act synergistically, resulting in an approximately 99% elimination of activated T cells at 10−8 mol/L (about 1.8 μg/mL). Because most natural killer (NK) cells are CD7+, NK activity was inhibited as well. Apart from the killing mediated by ricin A, binding of SPV-T3a by itself impaired in vitro cytotoxic T-cell cytotoxicity. Flow cytometric analysis revealed that this was due to both modulation of the CD3/T-cell receptor complex and activation-induced cell death. These results warranted evaluation of the IT combination in patients with refractory acute GVHD in an ongoing pilot study. So far, 4 patients have been treated with 3 to 4 infusions of 2 or 4 mg/m2 IT combination, administered intravenously at 48-hour intervals. The T1/2 was 6.7 hours, and peak serum levels ranged from 258 to 3210 ng/mL. Drug-associated side effects were restricted to limited edema, fever, and a modest rise of creatine kinase levels. One patient developed low-titer antibodies against ricin A. Infusions were associated with an immediate drop of circulating T cells, followed by a more gradual but continuing elimination of T/NK cells. One patient mounted an extensive CD8 T-cell response directly after treatment, not accompanied with aggravating GVHD. Two patients showed nearly complete remission of GVHD, despite unresponsiveness to the extensive pretreatment. These findings justify further investigation of the IT combination for treatment of diseases mediated by T cells.

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Interleukin-2 inhibits graft-versus-host disease-promoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects

Blood ◽

10.1182/blood.v83.9.2560.2560 ◽

1994 ◽

Vol 83 (9) ◽

pp. 2560-2569 ◽

Cited By ~ 67

Author(s):

M Sykes ◽

MW Harty ◽

GL Szot ◽

DA Pearson

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Interleukin 2 ◽

Cell Subset ◽

Spleen Cells ◽

Host Disease ◽

Graft Versus Host ◽

T Cell Subset ◽

Graft Versus Leukemia

Abstract We have recently shown that a short course of high-dose interleukin-2 (IL-2) can markedly inhibit the graft-versus-host disease (GVHD)- promoting activity of donor CD4+ T cells. The difficulty in dissociating GVHD-promoting from graft-versus-leukemia (GVL) effects of alloreactive donor T cells currently prevents clinical bone marrow transplantation (BMT) from fulfilling its full potential. To test the capacity of IL-2 treatment to promote such a dissociation, we have developed a new murine transplantable acute myelogenous leukemia model using a class II major histocompatibility complex-positive BALB/c Moloney murine leukemia virus-induced promonocytic leukemia, 2B-4–2. BALB/c mice receiving 2.5 x 10(5) 2B-4–2 cells intravenously 1 week before irradiation and syngeneic BMT died from leukemia within 2 to 4 weeks after BMT. Administration of syngeneic spleen cells and/or a 2.5- day course of IL-2 treatment alone did not inhibit leukemic mortality. In contrast, administration of non-T-cell-depleted fully allogeneic B10 (H-2b) spleen cells and T-cell-depleted B10 marrow led to a significant delay in leukemic mortality in IL-2-treated mice. In these animals GVHD was inhibited by IL-2 treatment. GVL effects were mediated entirely by donor CD4+ and CD8+ T cells. Remarkably, IL-2 administration did not diminish the magnitude of the GVL effect of either T-cell subset. This was surprising, because CD4-mediated GVHD was inhibited in the same animals in which CD4-mediated GVL effects were not reduced by IL-2 treatment. These results suggest a novel mechanism by which GVHD and GVL effects of a single unprimed alloreactive T-cell subset can be dissociated; different CD4 activities promote GVHD and GVL effects, and the former, but not the latter activities are inhibited by treatment with IL-2.

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Pretreatment of murine donor grafts with L-leucyl-L-leucine methyl ester: elimination of graft-versus-host disease without detrimental effects on engraftment

Blood ◽

10.1182/blood.v75.3.798.798 ◽

1990 ◽

Vol 75 (3) ◽

pp. 798-805 ◽

Cited By ~ 14

Author(s):

BR Blazar ◽

DL Thiele ◽

DA Vallera

Keyword(s):

Bone Marrow ◽

T Cell ◽

Methyl Ester ◽

Graft Versus Host Disease ◽

Host Cells ◽

Host Disease ◽

Murine Bone Marrow ◽

Graft Versus Host

Abstract Incubation of murine bone marrow and splenocytes with the dipeptide methyl ester, L-leucyl-L-leucine methyl ester (Leu-Leu-OMe), which results in the selective depletion of cytotoxic T cells and their precursors, natural killer cells, and monocytes, completely protected 30 recipients of fully allogeneic donor grafts from lethal graft-versus- host disease (GVHD). These results were comparable with those obtained in 30 recipients of anti-Thy 1.2 plus complement (C')-treated donor marrow. However, in contrast to antibody- and C'-dependent T-cell depletion, which reduces the level of donor cell engraftment in our model system, we did not observe such effects using Leu-Leu-OMe marrow pretreatment. As compared with the 24 H-2 typed recipients of anti-Thy 1.2 + C'-treated donor grafts, the 29 H-2 typed recipients of Leu-Leu- OMe-treated donor grafts had significantly (P less than .001) higher percentages of donor cells (mean = 93% v 74%) and significantly (P less than .001) lower percentages of host cells (mean = 6% v 15%) posttransplantation. In vitro limiting dilution assay (LDA) was performed to assess the comparative efficacy of cytolytic T-lymphocyte (CTL) precursor depletion by Leu-Leu-OMe or anti-Thy 1.2 + C' pretreatment. We observed greater levels of CTL precursor depletion in Leu-Leu-OMe treated as compared with anti-Thy 1.2 + C'-treated bone marrow plus spleen cells (BMS) obtained from nontransplanted mice. This suggests that the in vivo results cannot simply be attributed to a less efficacious functional inactivation of cytolytic T-cell precursors by Leu-Leu-OMe treatment as compared with anti-Thy 1.2 + C' treatment. Immunoreconstitution was similar in recipients of Leu-Leu-OMe-treated grafts and anti-Thy 1.2 + C'-treated grafts 100 days posttransplant. In our opinion, Leu-Leu-OMe marrow pretreatment deserves further investigation as a methodology to achieve GVHD prevention without significantly reducing the propensity toward host cell repopulation.

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Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice

Journal of Experimental Medicine ◽

10.1084/jem.20110466 ◽

2011 ◽

Vol 208 (12) ◽

pp. 2489-2496 ◽

Cited By ~ 59

Author(s):

Uri Sela ◽

Peter Olds ◽

Andrew Park ◽

Sarah J. Schlesinger ◽

Ralph M. Steinman

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Graft Versus Host Disease ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Spleen Cells ◽

Host Disease ◽

Graft Versus Host ◽

Naturally Occurring

Foxp3+ regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor β (TGF-β) and retinoic acid. We found that the CD11chigh dendritic cell fraction was the most potent at inducing high numbers of alloreactive Foxp3+ cells. The induced CD4+CD25+Foxp3+ cells appeared after extensive proliferation. When purified from the MLR, iT reg cells suppressed both primary and secondary MLR in vitro in an antigen-specific manner. After transfer into allogeneic mice, iT reg cells persisted for 6 mo and prevented graft versus host disease (GVHD) caused by co-transferred CD45RBhi T cells. Similar findings were made when iT reg cells were transferred after onset of GVHD. The CNS2 intronic sequence of the Foxp3 gene in the persisting iT reg cells was as demethylated as the corresponding sequence of naturally occurring T reg cells. These results indicate that induced Foxp3+ T reg cells, after proliferating and differentiating into antigen-specific suppressive T cells, can persist for long periods while suppressing a powerful inflammatory disease.

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Host γd T cells Exacerbate Experimental Acute Graft-Versus-Host Disease through Activation of Host Antigen Presenting Cells.

Blood ◽

10.1182/blood.v104.11.3045.3045 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3045-3045

Author(s):

Yoshinobu Maeda ◽

Pavan Reddy ◽

Chen Liu ◽

D. Keith Bishop ◽

James L.M. Ferrara

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Graft Versus Host Disease ◽

Serum Levels ◽

Host Disease ◽

Graft Versus Host ◽

Full Intensity

Abstract Large numbers of T cells bearing γd T cell receptors are present in graft-versus-host disease (GVHD) target tissues. We investigated the potential role of host γd T cells during acute GVHD in a well-characterized GVHD model following full intensity conditioning (11 Gy TBI). BM and spleen T cells from BALB/c (H2d) donors were transplanted into wild type (wt) B6, aß T cell deficient B6 (aß −/−) or γd T cell deficient B6 (γd −/−) hosts. γd −/− hosts demonstrated significantly better day 35 survival (85%) than wt (40%) or aß−/− hosts (18%) (P<0.05). Reconstitution of γd −/− B6 hosts with B6 type γd T cells 24 hr prior to BMT restored lethal GVHD (50 % day 35 survival). In vivo, γd −/− B6 hosts demonstrated at least a five fold reduction in donor T cell expansion and cytokine production. In vitro, T cells proliferated less when co-cultured with allogeneic γd −/− dendritic cells (DCs) than with wt DCs (40,127 ± 1634 vs. 72,503 ± 1296, P<0.05). BM-derived DCs cultured with γd T cells caused greater proliferation of allogeneic T cells than DCs cultured with aß T cells (15.1 ± 21 x 104 vs. 5.1 ± 1.2 x 104, P<0.05). We next tested the effect of γd T cells on host DCs in vivo using a model system in which only the DCs injected prior to BMT expressed the alloantigen that stimulated the GVHD reaction. MHC Class II −/− B6 mice that had been depleted of γd T cells were given 11 Gy TBI and injected one day prior to BMT with B6 DCs that had been co-cultured either with γd T cells or with medium. On day 0 both groups of recipient mice were injected with BM plus splenic T cells from allogeneic bm12 donors. On day +5, CD4+ donor T cells expanded four times more in recipients of DCs co-cultured with γd T cells than in recipients of control DCs and serum levels of TNF-a were significantly higher (36.7 + 6.8 vs. 21.3 + 3.7 pg/ml, P<0.05). Together these data demonstrate that γd T cells amplify the stimulatory function of host DCs and increase the severity of GVHD, suggesting that a new therapeutic target for the prevention of the major BMT toxicity.

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Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease

Blood ◽

10.1182/blood.v118.21.1972.1972 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1972-1972

Author(s):

Gerald P. Morris ◽

Geoffrey L Uy ◽

David L Donermeyer ◽

Paul M Allen ◽

John F. DiPersio

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Thymic Selection ◽

Cell Repertoire ◽

Host Disease ◽

Graft Versus Host

Abstract Abstract 1972 The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT. Disclosures: No relevant conflicts of interest to declare.

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Adoptive Transfer Of Ex Vivo “Educated” CD4+CD25+FoxP3+ Regulatory T Cells Effectively Treats Acute Graft Versus Host Disease Preserving Graft Versus Tumor Effect

Blood ◽

10.1182/blood.v122.21.4485.4485 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4485-4485

Author(s):

Antonio Pierini ◽

Dominik Schneidawind ◽

Mareike Florek ◽

Maite Alvarez ◽

Yuqiong Pan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Tumor Cells ◽

Graft Versus Host Disease ◽

Adoptive Transfer ◽

Cellular Therapy ◽

Ex Vivo ◽

Host Disease ◽

Graft Versus Host

Donor derived regulatory T cells (Tregs) effectively prevent graft versus host disease (GVHD) in mouse models and in early phase clinical trials. Interleukin 2 (IL-2) therapy in patients with chronic GVHD (cGVHD) can increase Treg number and the Treg/CD4+ T cell ratio resulting in organ damage reduction and symptom relief. Less is known regarding Treg-based treatment for acute GVHD (aGVHD). In this study we evaluated the role of donor Treg cellular therapy for aGVHD treatment in well established murine models. 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 luc+ Tcons. Naturally occurring CD4+CD25+FoxP3+ donor type Tregs (nTregs) were purified from C57BL/6 donor mice. 2.5x105/mouse nTregs were injected at day 6 or 7 after transplant in mice that showed clear clinical signs of aGVHD and Tcon proliferation assessed by bioluminescence imaging (BLI). Survival analysis showed a favorable trend for nTreg treated mice, but the impact of this treatment was modest and not statistically significant (p 0.08). aGVHD is a disease characterized by the activation and rapid proliferation of alloreactive donor conventional T cells (Tcons) directed against host antigens, so one of the major obstacles of this approach is to overcome the large number and effector function of activated Tcons. Several studies have utilized ex vivo expansion of Tregs to increase their number with the goal of maintaining suppressive function. We developed a different strategy with the intent to “educate” Tregs to specifically suppress the reactive Tcon population. We incubated 2.5x105 donor derived Tregs with irradiated (3000 cGy) blood of aGVHD affected mice for 20 hours without further stimulation and injected the entire pool of these cells, termed educated Treg (eTregs), at day 7 or 8 after transplant and Tcon injection. Interestingly eTregs significantly improved aGVHD affected mouse survival (p = 0.0025 vs Tcons alone). BLI showed no difference between the groups (p = 0.85) because the treatment intervened after Tcon proliferation and activation was initiated. To evaluate eTreg impact on graft versus tumor (GVT) effects, we transplanted BALB/C mice with C57BL/6 TCD BM and 1x104/mouse luc+ A20 tumor cells along with 1x106/mouse donor Tcons and 2.5x105 eTregs. Mice that received TCD BM and A20 tumor cells alone died from progressive tumor growth, while mice that received Tcons died from GVHD without tumor engraftment. Further animals that received both Tcon and eTreg treatment did not have tumor engraftment demonstrating that eTregs do not impact Tcon mediated GVT effects. Further studies are ongoing to characterize the eTreg population as compared to nTreg, with respect to expression of activation markers and in functional assays. Our observations indicate that Tregs can be ex vivo educated to suppress in vivo reactive and proliferating Tcons. Moreover our data demonstrate that eTreg adoptive transfer is clinically feasible and promising. These findings may be relevant for the development of clinical grade Treg based cellular therapy for the treatment of conditions caused by immune dysregulation such as aGVHD and autoimmune diseases and for transplant tolerance induction. 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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