Newborn blood can engraft adult mice without inducing graft-versus-host disease across non H-2 antigens

Cells derived from human cord blood were recently used instead of bone marrow (BM) for transplantation. However, several questions concerning the potential of these cells to reconstitute the hematopoietic and immunologic system of the recipient and to induce a graft-versus-host disease (GVHD) remain unanswered. Here we used newborn blood (NBB) cells from B10.D2 mice to engraft lethally irradiated (DBA/2 x B10.D2)F1 recipients incompatible for multiple non H-2 antigens. Few mature T cells were found in NBB as in adult BM and both contain around 10% to 20% SCA-1+ pluripotent progenitor cells. Yet numerous immature CD4+CD8+ TCR alpha/beta low Thy-1high T cells were present in NBB. In contrast, adult peripheral blood (PB) exhibits a mature T-cell phenotype and contains few progenitor cells. The injection of blood pooled from one to three newborn mice resulted in the engraftment of 71% to 86% of F1 recipients, which survived more than 100 days without clinical signs of GVHD. The injection of BM leads to 100% survival whereas the injection of adult PB resulted in rapid mortality, both without signs of GVHD. In NBB-engrafted F1 surviving mice, T-cell reconstitution preceded B-cell reconstitution, and the degree of donor cell chimerism increased progressively with time. Additionally, 2 to 4 months after transplantation, T cells from these mice were tolerant to host non H-2 antigens but kept reactivity against third-party Ags. Host specific tolerance in NBB-engrafted F1 mice was confirmed by in vivo transfer experiments. In conclusion, NBB cells were able to reconstitute the lymphohematopoietic system of lethally irradiated adult mice without inducing GVHD against incompatible non H-2 antigens. Thus, this experimental model in vivo may be relevant to the human cord blood transplantation.

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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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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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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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Prevention of Acute Graft-Versus-Host Disease Despite Compensatory Function of Lymphoid Organs In Vivo.

Blood ◽

10.1182/blood.v106.11.582.582 ◽

2005 ◽

Vol 106 (11) ◽

pp. 582-582 ◽

Cited By ~ 1

Author(s):

Andreas Beilhack ◽

Stephan Schulz ◽

Jeanette Baker ◽

Georg F. Beilhack ◽

Ryosei Nishimura ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Time Course ◽

Lymphoid Organs ◽

Host Disease ◽

Graft Versus Host ◽

Deficient Mice ◽

Acute Graft

Abstract Acute graft-versus-host disease (aGVHD) results from alloreactive donor derived T cells attacking targets in the gastrointestinal tract, liver and skin. We observed the initiation and rapid kinetics of aGVHD in a murine model [FVB/N (H-2q) into irradiated Balb/c (H-2d)] using in vivo bioluminescence imaging. The transition from the initiation to the effector phase of aGVHD (day 3–4) was characterized by rapid T cell proliferation and upregulation of gut homing receptors alpha4beta7, alphaEbeta7 and CCR9 on alloreactive T cells in Peyer’s patches (PP), mesenteric lymph nodes (LN) and spleen, but not peripheral LNs. Therefore we asked whether the lack of specific lymphoid priming sites would lead to decreased alloreactive T cell infiltration in the gut compared to the liver and skin. Using PP deficient mice, we observed that mesenteric LN and spleen compensate for the lack of PP as alloreactive priming sites. Transplantation of PP and LN deficient mice (TNFalpha-/-) showed that the spleen alone was sufficient to cause the complete profile of aGVHD with a time course similar to that of wildtype mice. Splenectomized mice with intact secondary lymphoid organs also developed aGVHD. Strikingly, treatment of splenectomized recipients with blocking antibodies against the lymphoid homing receptors L-selectin and MAdCAM-1 prevented GVHD with 100% survival (>120 d, p<0.0001). Our study shows that multiple priming sites are involved in GVHD initiation, the spleen compensating for the lack of PP and mesenteric LN, and vice versa. In contrast, splenectomy and antibody blocking resulted in a clear survival benefit for all recipients.

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Inhibition Of The Immunoproteasome Subunit LMP7 Decreases Alloresponses In Both MHC-Mismatched and miHA-Disparate Murine Models Of Graft Versus Host Disease

Blood ◽

10.1182/blood.v122.21.4472.4472 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4472-4472

Author(s):

Jennifer Matos ◽

Zheng Yang ◽

Eugenia Dziopa ◽

Leah Dziopa ◽

Christopher J. Kirk ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Research Funding ◽

Host Cells ◽

Mhc I ◽

Host Disease ◽

Graft Versus Host ◽

Drug Regimens

Background Proteasome inhibition has been studied and used as a therapeutic target in the treatment of autoimmune disorders and multiple myeloma. Immune system cells, especially antigen-presenting cells, express a higher basal level of immunoproteasomes, which are used to generate peptides that can be processed to fit in the groove of MHC class I molecules. ONX 0914 is a selective inhibitor of the immmunoproteasome that blocks LMP7-specific presentation of MHC-I restricted antigens, whereas PR-825 is a specific inhibitor of the b5 catalytic subunit generated by the constitutive proteasome. In previous work, we found that ONX 0914 administration early after transplantation significantly improved the survival of recipient mice in an MHC-matched minor histocompatibility antigen (miHA)-disparate (B10.BR -> CBA, lethally irradiated) murine model of graft-versus-host disease (GVHD). To further elucidate the mechanism of action, we compared alloreactive responses via IFN-g production in mixed lymphocyte reactions (MLR) in which stimulator cells or responder T cells were pretreated with either ONX 0914 or PR-825. MLR were conducted for the B10.BR anti-C56BL/6y (B6) MHC-mismatched, the CD8+ T cell-mediated B10.BR anti-CBA, and the CD4+ T cell-mediated B6 anti-BALB.B MHC-matched/ miHA-disparate strain combinations. Methods Stimulator splenocytes were treated for one hour with ONX 0914 (300 nM) or PR-825 (125 nM) before or immediately after exposure to irradiation (30 Gy) prior to initiation of the MLR. In addition, to try to optimize putative in vivo drug regimens, we determined the timing of release/degradation of antigenic peptides presented in the context of MHC-I. To this end, the MC57G fibrosarcoma cell line was transiently transfected with a GFP tagged SIINFEKL plasmid, cells were treated with different drug regimens and exposure times of ONX 0914 or PR-825, and monitored over 48 hours for surface expression of MHC-I presented SIINFEKL peptide using flow cytometry. Results ELISpot assays showed a statistically significant decrease in the number of IFN-g producing cells when stimulators (B6 splenocytes) were pretreated with ONX 0914 compared to pretreated responder (B10.BR) cells (60.21% vs. 1.75% respectively, p< 0.01). In the miHA disparate combinations, the percentage decrease in IFN-g+ spots was ∼30% when stimulators were treated with ONX 0914, whereas only a ∼15% decrease was observed with PR-825 pretreatment. Furthermore, IFN-g production was not dependent upon the timing of exposure to irradiation, as ELISpot counts were equally decreased when drug pretreatments were performed before irradiation or just after exposure. Antigenic peptide presentation was maximally decreased in transfected MC57G cells 48 h after treatment (i.e., SIINFEKL % in GFP+ MC57G cells treated with DMSO was equal to 83% vs. 76% when cells were exposed to ONX 0914 [300 nM, for 24 h]. This result suggests that for in vivo application, pretreatment of recipient mice with ONX 0914 to decrease miHA presentation by host cells (24-72 hours prior to bone marrow transplantation) may provide further amelioration of GVHD development. Conclusion Taken together, these data suggest that downregulation of LMP7-mediated presentation of MHC-I restricted antigens by host cells likely modulated stimulation and IFN-g production of donor T cells in vivo, rather than acting on effector cells directly, and accounted in part for the improved survival rate experienced by recipient mice treated with ONX 0914. Disclosures: Matos: Onyx Pharmaceuticals: Research Funding. Dziopa:Onyx Pharmaceuticals: Research Funding. Kirk:Onyx Pharmaceuticals: Employment. Korngold:Onyx Pharmaceuticals: Research Funding. Zilberberg:Onyx Pharmaceuticals: Research Funding.

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In vivo analyses of early events in acute graft-versus-host disease reveal sequential infiltration of T-cell subsets

Blood ◽

10.1182/blood-2005-02-0509 ◽

2005 ◽

Vol 106 (3) ◽

pp. 1113-1122 ◽

Cited By ~ 229

Author(s):

Andreas Beilhack ◽

Stephan Schulz ◽

Jeanette Baker ◽

Georg F. Beilhack ◽

Courtney B. Wieland ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Lymphoid Organs ◽

T Cell Subsets ◽

Effector Memory ◽

Host Disease ◽

Graft Versus Host ◽

Secondary Lymphoid Organs

AbstractGraft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (TEM) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.

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In vivo dynamics of regulatory T-cell trafficking and survival predict effective strategies to control graft-versus-host disease following allogeneic transplantation

Blood ◽

10.1182/blood-2006-08-044529 ◽

2006 ◽

Vol 109 (6) ◽

pp. 2649-2656 ◽

Cited By ~ 154

Author(s):

Vu H. Nguyen ◽

Robert Zeiser ◽

Daniel L. daSilva ◽

Daisy S. Chang ◽

Andreas Beilhack ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Lymphoid Organs ◽

Allogeneic Bone ◽

Major Barrier ◽

Peripheral Tissues ◽

Host Disease ◽

Graft Versus Host

Abstract CD4+CD25+ regulatory T cells (Tregs) suppress immune responses to alloantigens. The in vivo circulation and tissue localization of Tregs during an adaptive immune response remain unclear. We noninvasively tracked luciferase-expressing Tregs over time in an allogeneic bone marrow transplant model and demonstrated colocalization with effector T cells and initial expansion in secondary lymphoid organs before migration into inflamed tissues. Inflammation induced by irradiation and the allogeneic setting provided crucial stimuli for early Treg expansion and migration, leading to parallel reduction of effector T-cell proliferation in lymphoid organs and peripheral tissues. Treg transplants conferred long-term protection from systemic inflammatory challenge consistent with Treg in vivo survival. Suppression occurred during multiple phases of inflammation, but is optimal in the initial phase, providing protection from graft-versus-host disease while maintaining the graft-versus-tumor effect even at physiologic doses of Tregs due to their in vivo expansion, hence overcoming a major barrier to potential clinical applications of Tregs given their rarity.

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Attenuation of graft-versus-host disease and graft rejection by ex vivo immunotoxin elimination of alloreactive T cells in an H-2 haplotype disparate mouse combination

Blood ◽

10.1182/blood.v83.1.288.bloodjournal831288 ◽

1994 ◽

Vol 83 (1) ◽

pp. 288-298 ◽

Cited By ~ 1

Author(s):

M Cavazzana-Calvo ◽

JL Stephan ◽

S Sarnacki ◽

S Chevret ◽

C Fromont ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Graft Rejection ◽

Ex Vivo ◽

Interleukin 2 ◽

In Vivo Model ◽

Host Disease ◽

Graft Versus Host

A mouse anti-interleukin-2 receptor A-chain-specific PC61-immunotoxin (PC61-IT) strongly inhibited a primary mixed lymphocyte culture and major histocompatibility complex (MHC)-restricted cytotoxicity. The allodepleted T cells retained their proliferative and cytotoxic capacities in response to third-party stimulation, showing that PC61-IT specifically deleted recipient antigen-specific T-cell clones from the donor mouse. The ability of this specific allodepletion to prevent graft-versus-host disease (GVHD) and graft rejection was investigated in vivo. IT-depleted, activated parental T lymphocytes (C3H/eB) were intravenously injected into lethally irradiated CDF1 mice. GVHD was evaluated after 6 days on the severity of gut lesions. PC61-IT-treated cells significantly reduced both donor T-cell infiltration and acceleration of epithelial renewal (a sensitive index of gut damage) as compared with those for the corresponding untreated controls. The effect of selective allo-depletion on prevention of GVHD and graft rejection was further studied after MHC-haploincompatible bone marrow (BM) transplantation. A significant increase in survival was observed in mice receiving 2 x 10(6) T-cell-depleted BM cells and 0.5 x 10(6) PC61-IT-treated T cells, because one-third were alive without GVHD (and with stable full or partial engraftment) after 100 days, whereas all the mice infused with BM and sham-treated T cells died within 80 days from GVHD, and all the mice infused with BM cells alone rejected grafts. Furthermore, specific tolerance in chimeras towards donor cells could be shown. These results as observed in an experimental in vivo model corroborate previous results obtained in vitro in humans and lead us to consider the use of this selective allodepletion in human BM transplant from donors other than identical familial siblings.

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STAT-3 and ERK 1/2 phosphorylation are critical for T-cell alloactivation and graft-versus-host disease

Blood ◽

10.1182/blood-2008-03-147322 ◽

2008 ◽

Vol 112 (13) ◽

pp. 5254-5258 ◽

Cited By ~ 47

Author(s):

Sydney X. Lu ◽

Onder Alpdogan ◽

Janine Lin ◽

Robert Balderas ◽

Roberto Campos-Gonzalez ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Drug Targets ◽

Allogeneic Bone ◽

Specific Flow ◽

Host Disease ◽

Graft Versus Host

Abstract Graft-versus-host disease (GVHD) is a serious complication of allogeneic bone marrow transplantation, and donor T cells are indispensable for GVHD. Current therapies have limited efficacy, selectivity, and high toxicities. We used a novel flow cytometry technique for the analysis of intracellular phosphorylation events in single cells in murine BMT models to identify and validate novel GVHD drug targets.1-7 This method circumvents the requirement for large numbers of purified cells, unlike western blots. We defined a signaling profile for alloactivated T cells in vivo and identified the phosphorylation of ERK1/2 and STAT-3 as important events during T-cell (allo)activation in GVHD. We establish that interference with STAT-3 phosphorylation can inhibit T-cell activation and proliferation in vitro and GVHD in vivo. This suggests that phospho-specific flow cytometry is useful for the identification of promising drug targets, and ERK1/2 and STAT-3 phosphorylation in alloactivated T cells may be important for GVHD.

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